21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

Advances in DNA metabarcoding for food and wildlife forensic species identification.

PubMed

Staats, Martijn; Arulandhu, Alfred J; Gravendeel, Barbara; Holst-Jensen, Arne; Scholtens, Ingrid; Peelen, Tamara; Prins, Theo W; Kok, Esther

2016-07-01

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

PubMed

Versteirt, V; Nagy, Z T; Roelants, P; Denis, L; Breman, F C; Damiens, D; Dekoninck, W; Backeljau, T; Coosemans, M; Van Bortel, W

2015-03-01

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species. © 2014 John Wiley & Sons Ltd.

Forensic identification of CITES protected slimming cactus (Hoodia) using DNA barcoding.

PubMed

Gathier, Gerard; van der Niet, Timotheus; Peelen, Tamara; van Vugt, Rogier R; Eurlings, Marcel C M; Gravendeel, Barbara

2013-11-01

Slimming cactus (Hoodia), found only in southwestern Africa, is a well-known herbal product for losing weight. Consequently, Hoodia extracts are sought-after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA-trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI-MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade. © 2013 American Academy of Forensic Sciences.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Identification of Amazonian trees with DNA barcodes.

PubMed

Gonzalez, Mailyn Adriana; Baraloto, Christopher; Engel, Julien; Mori, Scott A; Pétronelli, Pascal; Riéra, Bernard; Roger, Aurélien; Thébaud, Christophe; Chave, Jérôme

2009-10-16

Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests. Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon. We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs.

Identification of Amazonian Trees with DNA Barcodes

PubMed Central

Gonzalez, Mailyn Adriana; Baraloto, Christopher; Engel, Julien; Mori, Scott A.; Pétronelli, Pascal; Riéra, Bernard; Roger, Aurélien; Thébaud, Christophe; Chave, Jérôme

2009-01-01

Background Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests. Methodology/Principal Findings Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon. Conclusion/Significance We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs. PMID:19834612

The changing epitome of species identification – DNA barcoding

PubMed Central

Ajmal Ali, M.; Gyulai, Gábor; Hidvégi, Norbert; Kerti, Balázs; Al Hemaid, Fahad M.A.; Pandey, Arun K.; Lee, Joongku

2014-01-01

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The ‘DNA barcodes’ show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more. PMID:24955007

Identification of species with DNA-based technology: current progress and challenges.

PubMed

Pereira, Filipe; Carneiro, João; Amorim, António

2008-01-01

One of the grand challenges of modern biology is to develop accurate and reliable technologies for a rapid screening of DNA sequence variation. This topic of research is of prime importance for the detection and identification of species in numerous fields of investigation, such as taxonomy, epidemiology, forensics, archaeology or ecology. Molecular identification is also central for the diagnosis, treatment and control of infections caused by different pathogens. In recent years, a variety of DNA-based approaches have been developed for the identification of individuals in a myriad of taxonomic groups. Here, we provide an overview of most commonly used assays, with emphasis on those based on DNA hybridizations, restriction enzymes, random PCR amplifications, species-specific PCR primers and DNA sequencing. A critical evaluation of all methods is presented focusing on their discriminatory power, reproducibility and user-friendliness. Having in mind that the current trend is to develop small-scale devices with a high-throughput capacity, we briefly review recent technological achievements for DNA analysis that offer great potentials for the identification of species.

[Application of mtDNA polymorphism in species identification of sarcosaphagous insects].

PubMed

Li, Xiang; Cai, Ji-feng

2011-04-01

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.

A grass molecular identification system for forensic botany: a critical evaluation of the strengths and limitations.

PubMed

Ward, Jodie; Gilmore, Simon R; Robertson, James; Peakall, Rod

2009-11-01

Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA-based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single-nucleotide polymorphisms were utilized as molecular markers for subfamily to species-level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated "proof of concept" of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

Identification of Rays through DNA Barcoding: An Application for Ecologists

PubMed Central

Cerutti-Pereyra, Florencia; Meekan, Mark G.; Wei, Nu-Wei V.; O'Shea, Owen; Bradshaw, Corey J. A.; Austin, Chris M.

2012-01-01

DNA barcoding potentially offers scientists who are not expert taxonomists a powerful tool to support the accuracy of field studies involving taxa that are diverse and difficult to identify. The taxonomy of rays has received reasonable attention in Australia, although the fauna in remote locations such as Ningaloo Reef, Western Australia is poorly studied and the identification of some species in the field is problematic. Here, we report an application of DNA-barcoding to the identification of 16 species (from 10 genera) of tropical rays as part of an ecological study. Analysis of the dataset combined across all samples grouped sequences into clearly defined operational taxonomic units, with two conspicuous exceptions: the Neotrygon kuhlii species complex and the Aetobatus species complex. In the field, the group that presented the most difficulties for identification was the spotted whiptail rays, referred to as the ‘uarnak’ complex. Two sets of problems limited the successful application of DNA barcoding: (1) the presence of cryptic species, species complexes with unresolved taxonomic status and intra-specific geographical variation, and (2) insufficient numbers of entries in online databases that have been verified taxonomically, and the presence of lodged sequences in databases with inconsistent names. Nevertheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to highlight species complexes where taxonomic uncertainty might confound ecological data. PMID:22701556

Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive.

PubMed

Hering, Daniel; Borja, Angel; Jones, J Iwan; Pont, Didier; Boets, Pieter; Bouchez, Agnes; Bruce, Kat; Drakare, Stina; Hänfling, Bernd; Kahlert, Maria; Leese, Florian; Meissner, Kristian; Mergen, Patricia; Reyjol, Yorick; Segurado, Pedro; Vogler, Alfried; Kelly, Martyn

2018-07-01

Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification

Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Y.; Dunn, J.; Gao, S.

2008-10-31

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing asmore » little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.« less

Identification of body fluid-specific DNA methylation markers for use in forensic science.

PubMed

Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

PubMed

Shokralla, Shadi; Hellberg, Rosalee S; Handy, Sara M; King, Ian; Hajibabaei, Mehrdad

2015-10-30

Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences-mini-barcodes-for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127-314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

BOLDMirror: a global mirror system of DNA barcode data.

PubMed

Liu, D; Liu, L; Guo, G; Wang, W; Sun, Q; Parani, M; Ma, J

2013-11-01

DNA barcoding is a novel concept for taxonomic identification using short, specific genetic markers and has been applied to study a large number of eukaryotes. The huge amount of data output generated by DNA barcoding requires well-organized information systems. Besides the Barcode of Life Data system (BOLD) established in Canada, the mirror system is also important for the international barcode of life project (iBOL). For this purpose, we developed the BOLDMirror, a global mirror system of DNA barcode data. It is open-sourced and can run on the LAMP (Linux + Apache + MySQL + PHP) environment. BOLDMirror has data synchronization, data representation and statistics modules, and also provides spaces to store user operation history. BOLDMirror can be accessed at http://www.boldmirror.net and several countries have used it to setup their site of DNA barcoding. © 2012 John Wiley & Sons Ltd.

Logical Framework of Forensic Identification: Ability to Resist Fabricated DNA.

PubMed

Wang, Zheng; Zhou, Di; Zhang, Suhua; Bian, Yingnan; Hu, Zhen; Zhu, Ruxin; Lu, Daru; Li, Chengtao

2015-12-01

Over the past 30 years, DNA analysis has revolutionized forensic science and has become the most useful single tool in the multifaceted fight against crime. Today, DNA profiling with sets of highly polymorphic autosomal short tandem repeat markers is widely employed and accepted in the courts due to its high discriminating power and reliability. However, an artificial bloodstain purposefully created using molecular biology techniques succeeded in tricking a leading forensic DNA laboratory. The disturbing possibility that a forensic DNA profile can be faked shocked the general public and the mass media, and generated serious discussion about the credibility of DNA evidence. Herein, we present two exemplary assays based on tissue-specific methylation patterns and cell-specific mRNA expression, respectively. These two assays can be integrated into the DNA analysis pipelines without consumption of additional samples. We show that the two assays can not only distinguish between artificial and genuine samples, but also provide information on tissue origin. The two assays were tested on natural and artificial bloodstains (generated by polymerase chain reaction and whole genome amplification technique) and the results illustrated that the logical framework of forensic identification is still useful for forensic identification with the high credibility.

DNA methods for identification of Chinese medicinal materials

PubMed Central

Yip, Pui Ying; Chau, Chi Fai; Mak, Chun Yin; Kwan, Hoi Shan

2007-01-01

As adulterated and substituted Chinese medicinal materials are common in the market, therapeutic effectiveness of such materials cannot be guaranteed. Identification at species-, strain- and locality-levels, therefore, is required for quality assurance/control of Chinese medicine. This review provides an informative introduction to DNA methods for authentication of Chinese medicinal materials. Technical features and examples of the methods based on sequencing, hybridization and polymerase chain reaction (PCR) are described and their suitability for different identification objectives is discussed. PMID:17803808

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

PubMed Central

Hong, Ka Lok

2015-01-01

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

PubMed

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

PubMed Central

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

PubMed

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

A laboratory information management system for DNA barcoding workflows.

PubMed

Vu, Thuy Duong; Eberhardt, Ursula; Szöke, Szániszló; Groenewald, Marizeth; Robert, Vincent

2012-07-01

This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA sequence markers. We aim at generating barcode data for all strains (or specimens) included in the collection (currently ca. 80 k). The LIMS has been developed to better manage large amounts of sequence data and to keep track of the whole experimental procedure. The system has allowed us to classify strains more efficiently as the quality of sequence data has improved, and as a result, up-to-date taxonomic names have been given to strains and more accurate correlation analyses have been carried out.

Characteristics of Populations of the Russian Federation over the Panel of Fifteen Loci Used for DNA Identification and in Forensic Medical Examination

PubMed Central

A Stepanov, V.; Balanovsky, O.P.; Melnikov, A.V.; Lash-Zavada, A.Yu.; Khar’kov, V.N.; Tyazhelova, T.V.; Akhmetova, V.L.; Zhukova, O.V.; Shneider, Yu.V.; Shil’nikova, I.N.; Borinskaya, S.A.; Marusin, A.V.; Spiridonova, M.G.; Simonova, K.V.; Khitrinskaya, I.Yu.; Radzhabov, M.O.; Romanov, A.G.; Shtygasheva, O.V.; Koshel’, S.M.; Balanovskaya, E.V.; Rybakova, A.V.; Khusnutdinova, E.K.; Puzyrev, V.P.; Yankovsky, N.K.

2011-01-01

Seventeen population groups within the Russian Federation were characterized for the first time using a panel of 15 genetic markers that are used for DNA identification and in forensic medical examinations. The degree of polymorphism and population diversity of microsatellite loci within the Power Plex system (Promega) in Russian populations; the distribution of alleles and genotypes within the populations of six cities and 11 ethnic groups of the Russian Federation; the levels of intra- and interpopulation genetic differentiation of population; genetic relations between populations; and the identification and forensic medical characteristics of the system of markers under study were determined. Significant differences were revealed between the Russian populations and the U.S. reference base that was used recently in the forensic medical examination of the RF. A database of the allelic frequencies of 15 microsatellite loci that are used for DNA identification and forensic medical examination was created; the database has the potential of becoming the reference for performing forensic medical examinations in Russia. The spatial organization of genetic diversity over the panel of the STR markers that are used for DNA identification was revealed. It represents the general regularities of geographical clusterization of human populations over various types of genetic markers. The necessity to take into account a population’s genetic structure during forensic medical examinations and DNA identification of criminal suspects was substantiated. PMID:22649684

Choosing relatives for DNA identification of missing persons.

PubMed

Ge, Jianye; Budowle, Bruce; Chakraborty, Ranajit

2011-01-01

DNA-based analysis is integral to missing person identification cases. When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. Generally, more reference relatives render greater accuracy of identification. However, it is costly to type multiple references. Thus, at times, decisions may need to be made on which relatives to type. In this study, pedigrees for 37 common reference scenarios with 13 CODIS STRs were simulated to rank the information content of different combinations of relatives. The results confirm that first-order relatives (parents and fullsibs) are the most preferred relatives to identify missing persons; fullsibs are also informative. Less genetic dependence between references provides a higher on average likelihood ratio. Distant relatives may not be helpful solely by autosomal markers. But lineage-based Y chromosome and mitochondrial DNA markers can increase the likelihood ratio or serve as filters to exclude putative relationships. © 2010 American Academy of Forensic Sciences.

Potential for DNA-based identification of Great Lakes fauna: Match and mismatch between taxa inventories and DNA barcode libraries

EPA Science Inventory

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to biotic condition assessment and non-native species early-detection monitoring. However, the abi...

Assessment of three plastid DNA barcode markers for identification of Clinacanthus nutans (Acanthaceae).

PubMed

Ismail, Noor Zafirah; Arsad, Hasni; Samian, Mohammed Razip; Hamdan, Mohammad Razak; Othman, Ahmad Sofiman

2018-01-01

This study was conducted to determine the feasibility of using three plastid DNA regions ( matK , trnH - psbA , and rbcL ) as DNA barcodes to identify the medicinal plant Clinacanthus nutans . In this study, C. nutans was collected at several different locations. Total genomic DNA was extracted, amplified by polymerase chain reaction (PCR), and sequenced using matK , trnH - psbA , and rbcL , primers. DNA sequences generated from PCR were submitted to the National Center for Biotechnology Information's (NCBI) GenBank. Identification of C. nutans was carried out using NCBI's Basic Local Alignment Search Tool (BLAST). The rbcL and trnH - psbA regions successfully identified C. nutans with sequencing rates of 100% through BLAST identification. Molecular Evolutionary Genetics Analysis (MEGA) 6.0 was used to analyze interspecific and intraspecific divergence of plastid DNA sequences. rbcL and matK exhibited the lowest average interspecific distance (0.0487 and 0.0963, respectively), whereas trnH - psbA exhibited the highest average interspecific distance (0.2029). The R package Spider revealed that trnH - psbA correctly identified Barcode of Life Data System (BOLD) 96%, best close match 79%, and near neighbor 100% of the species, compared to matK (BOLD 72%; best close match 64%; near neighbor 78%) and rbcL (BOLD 77%; best close match 62%; near neighbor 88%). These results indicate that trnH - psbA is very effective at identifying C. nutans , as it performed well in discriminating species in Acanthaceae.

The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair.

PubMed

Gruszka, Damian; Marzec, Marek; Szarejko, Iwona

2012-06-14

The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants. These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the "Barley Genome version 0.05" database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair. The presented database provides information about the Arabidopsis genes involved in DNA replication and repair, their expression patterns and models

How effective are DNA barcodes in the identification of African rainforest trees?

PubMed

Parmentier, Ingrid; Duminil, Jérôme; Kuzmina, Maria; Philippe, Morgane; Thomas, Duncan W; Kenfack, David; Chuyong, George B; Cruaud, Corinne; Hardy, Olivier J

2013-01-01

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95-100% success), but less for species identification (71-88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84-90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.

DNA Identification of Skeletal Remains from World War II Mass Graves Uncovered in Slovenia

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Bakal, Narcisa; Haverić, Sanin; Kalamujić, Belma; Kovačević, Lejla; Ramić, Jasmin; Pojskić, Naris; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Hadžiselimović, Rifat; Drobnič, Katja; Huffine, Ed; Davoren, Jon; Primorac, Dragan

2007-01-01

Aim To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Škofja Loka, Slovenia. Methods The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. Results Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. Conclusion Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project. PMID:17696306

DNA-based identification of Brassica vegetable species for the juice industry.

PubMed

Etoh, Kazumi; Niijima, Noritaka; Yokoshita, Masahiko; Fukuoka, Shin-Ichi

2003-10-01

Since kale (Brassica oleracea var. acephala), a cruciferous vegetable with a high level of vitamins and functional compounds beneficial to health and wellness, has become widely used in the juice industry, a precise method for quality control of vegetable species is necessary. We describe here a DNA-based identification method to distinguish kale from cabbage (Brassica oleracea var. capitata), a closely related species, which can be inadvertently mixed with kale during the manufacturing process. Using genomic DNA from these vegetables and combinatory sets of nucleotide primers, we screened for random amplified polymorphic DNA (RAPD) fragments and found three cabbage-specific fragments. These RAPD fragments, with lengths of 1.4, 0.5, and 1.5 kb, were purified, subcloned, and sequenced. Based on sequence-tagged sites (STS), we designed sets of primers to detect cabbage-specific identification (CAI) DNA markers. Utilizing the CAI markers, we successfully distinguished more than 10 different local cabbage accessions from 20 kale accessions, and identified kale juices experimentally spiked with different amounts of cabbage.

DNA identification of human remains in Disaster Victim Identification (DVI): An efficient sampling method for muscle, bone, bone marrow and teeth.

PubMed

de Boer, Hans H; Maat, George J R; Kadarmo, D Aji; Widodo, Putut T; Kloosterman, Ate D; Kal, Arnoud J

2018-06-04

In disaster victim identification (DVI), DNA profiling is considered to be one of the most reliable and efficient means to identify bodies or separated body parts. This requires a post mortem DNA sample, and an ante mortem DNA sample of the presumed victim or their biological relative(s). Usually the collection of an adequate ante mortem sample is technically simple, but the acquisition of a good quality post mortem sample under unfavourable DVI circumstances is complicated due to the variable degree of preservation of the human remains and the high risk of DNA (cross) contamination. This paper provides the community with an efficient method to collect post-mortem DNA samples from muscle, bone, bone marrow and teeth, with a minimal risk of contamination. Our method has been applied in a recent, challenging DVI operation (i.e. the identification of the 298 victims of the MH17 airplane crash in 2014). 98,2% of the collected PM samples provided the DVI team with highly informative DNA genotyping results without the risk of contamination and consequent mistyping the victim's DNA. Moreover, the method is easy, cheap and quick. This paper provides the DVI community with a step-wise instructions with recommendations for the type of tissue to be sampled and the site of excision (preferably the upper leg). Although initially designed for DVI purposes, the method is also suited for the identification of individual victims. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

PubMed

Ricci, Ugo; Sani, Ilaria; Klintschar, Michael; Cerri, Nicoletta; De Ferrari, Francesco; Giovannucci Uzielli, Maria Luisa

2003-06-01

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

DNA barcoding for molecular identification of Demodex based on mitochondrial genes.

PubMed

Hu, Li; Yang, YuanJun; Zhao, YaE; Niu, DongLing; Yang, Rui; Wang, RuiLing; Lu, Zhaohui; Li, XiaoQi

2017-12-01

There has been no widely accepted DNA barcode for species identification of Demodex. In this study, we attempted to solve this issue. First, mitochondrial cox1-5' and 12S gene fragments of Demodex folloculorum, D. brevis, D. canis, and D. caprae were amplified, cloned, and sequenced for the first time; intra/interspecific divergences were computed and phylogenetic trees were reconstructed. Then, divergence frequency distribution plots of those two gene fragments were drawn together with mtDNA cox1-middle region and 16S obtained in previous studies. Finally, their identification efficiency was evaluated by comparing barcoding gap. Results indicated that 12S had the higher identification efficiency. Specifically, for cox1-5' region of the four Demodex species, intraspecific divergences were less than 2.0%, and interspecific divergences were 21.1-31.0%; for 12S, intraspecific divergences were less than 1.4%, and interspecific divergences were 20.8-26.9%. The phylogenetic trees demonstrated that the four Demodex species clustered separately, and divergence frequency distribution plot showed that the largest intraspecific divergence of 12S (1.4%) was less than cox1-5' region (2.0%), cox1-middle region (3.1%), and 16S (2.8%). The barcoding gap of 12S was 19.4%, larger than cox1-5' region (19.1%), cox1-middle region (11.3%), and 16S (13.0%); the interspecific divergence span of 12S was 6.2%, smaller than cox1-5' region (10.0%), cox1-middle region (14.1%), and 16S (11.4%). Moreover, 12S has a moderate length (517 bp) for sequencing at once. Therefore, we proposed mtDNA 12S was more suitable than cox1 and 16S to be a DNA barcode for classification and identification of Demodex at lower category level.

Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fields, C.A.

1994-09-01

This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

Using DNA chips for identification of tephritid pest species.

PubMed

Chen, Yen-Hou; Liu, Lu-Yan; Tsai, Wei-Huang; Haymer, David S; Lu, Kuang-Hui

2014-08-01

The ability correctly to identify species in a rapid and reliable manner is critical in many situations. For insects in particular, the primary tools for such identification rely on adult-stage morphological characters. For a number of reasons, however, there is a clear need for alternatives. This paper reports on the development of a new method employing DNA biochip technology for the identification of pest species within the family Tephritidae. The DNA biochip developed and tested here quickly and efficiently identifies and discriminates between several tephritid species, except for some that are members of a complex of closely related taxa and that may in fact not represent distinct biological species. The use of these chips offers a number of potential advantages over current methods. Results can be obtained in less than 5 h using material from any stage of the life cycle and with greater sensitivity than other methods currently available. This technology provides a novel tool for the rapid and reliable identification of several major pest species that may be intercepted in imported fruits or other commodities. The existing chips can also easily be expanded to incorporate additional markers and species as needed. © 2013 Society of Chemical Industry.

DNA Barcoding of Neotropical Sand Flies (Diptera, Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

PubMed Central

Pinto, Israel de Souza; Chagas, Bruna Dias das; Rodrigues, Andressa Alencastre Fuzari; Ferreira, Adelson Luiz; Rezende, Helder Ricas; Bruno, Rafaela Vieira; Falqueto, Aloisio; Andrade-Filho, José Dilermando; Galati, Eunice Aparecida Bianchi; Shimabukuro, Paloma Helena Fernandes; Brazil, Reginaldo Peçanha

2015-01-01

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil. PMID:26506007

DNA Barcoding of Neotropical Sand Flies (Diptera, Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil.

PubMed

Pinto, Israel de Souza; Chagas, Bruna Dias das; Rodrigues, Andressa Alencastre Fuzari; Ferreira, Adelson Luiz; Rezende, Helder Ricas; Bruno, Rafaela Vieira; Falqueto, Aloisio; Andrade-Filho, José Dilermando; Galati, Eunice Aparecida Bianchi; Shimabukuro, Paloma Helena Fernandes; Brazil, Reginaldo Peçanha; Peixoto, Alexandre Afranio

2015-01-01

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23-19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.

Results of a collaborative study on DNA identification of aged bone samples

PubMed Central

Vanek, Daniel; Budowle, Bruce; Dubska-Votrubova, Jitka; Ambers, Angie; Frolik, Jan; Pospisek, Martin; Al Afeefi, Ahmed Anwar; Al Hosani, Khalid Ismaeil; Allen, Marie; Al Naimi, Khudooma Saeed; Al Salafi, Dina; Al Tayyari, Wafa Ali Rashid; Arguetaa, Wendy; Bottinelli, Michel; Bus, Magdalena M.; Cemper-Kiesslich, Jan; Cepil, Olivier; De Cock, Greet; Desmyter, Stijn; El Amri, Hamid; El Ossmani, Hicham; Galdies, Ruth; Grün, Sebastian; Guidet, Francois; Hoefges, Anna; Iancu, Cristian Bogdan; Lotz, Petra; Maresca, Alessandro; Nagy, Marion; Novotny, Jindrich; Rachid, Hajar; Rothe, Jessica; Stenersen, Marguerethe; Stephenson, Mishel; Stevanovitch, Alain; Strien, Juliane; Sumita, Denilce R.; Vella, Joanna; Zander, Judith

2017-01-01

Aim A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. Methods Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. Results Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. Conclusion The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized. PMID:28613037

How Effective Are DNA Barcodes in the Identification of African Rainforest Trees?

PubMed Central

Parmentier, Ingrid; Duminil, Jérôme; Kuzmina, Maria; Philippe, Morgane; Thomas, Duncan W.; Kenfack, David; Chuyong, George B.; Cruaud, Corinne; Hardy, Olivier J.

2013-01-01

Background DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. Methodology/Principal Findings We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95–100% success), but less for species identification (71–88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84–90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. Conclusions/Significance Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements

Aspergillus and Penicillium identification using DNA sequences: Barcode or MLST?

USDA-ARS?s Scientific Manuscript database

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through...

Identification of the skeletal remains of a murder victim by DNA analysis.

PubMed

Hagelberg, E; Gray, I C; Jeffreys, A J

1991-08-01

There is considerable anthropological and forensic interest in the possibility of DNA typing skeletal remains. Trace amounts of DNA can be recovered even from 5,500-year-old bones and multicopy human mitochondrial DNA sequences can frequently be amplified from such DNA using the polymerase chain reaction (PCR). But given the sensitivity of PCR, it is very difficult to exclude contaminating material. We now report the successful identification of the 8-year-old skeletal remains of a murder victim, by comparative typing of nuclear microsatellite markers in the remains and in the presumptive parents of the victim. This analysis establishes the authenticity of the bone DNA and the feasibility of bone DNA typing in forensic investigations.

DNA Barcoding of Malagasy Rosewoods: Towards a Molecular Identification of CITES-Listed Dalbergia Species.

PubMed

Hassold, Sonja; Lowry, Porter P; Bauert, Martin R; Razafintsalama, Annick; Ramamonjisoa, Lolona; Widmer, Alex

2016-01-01

Illegal selective logging of tropical timber is of increasing concern worldwide. Madagascar is a biodiversity hotspot and home to some of the world's most sought after tropical timber species. Malagasy rosewoods belong to the genus Dalbergia (Fabaceae), which is highly diverse and has a pantropical distribution, but these timber species are among the most threatened as a consequence of intensive illegal selective logging and deforestation. Reliable identification of Dalbergia species from Madagascar is important for law enforcement but is almost impossible without fertile plant material, which is often unavailable during forest inventories or when attempting to identify logged trees of cut wood. DNA barcoding has been promoted as a promising tool for species identification in such cases. In this study we tested whether DNA barcoding with partial sequences of three plastid markers (matK, rbcL and trnL (UAA)) can distinguish between Dalbergia from Madagascar and from other areas of its distributional range, and whether Malagasy species can be distinguished from one another. Phylogenetic analyses revealed that the Malagasy Dalbergia species studied form two monophyletic groups, each containing two subgroups, only one of which corresponds to a single species. We characterized diagnostic polymorphisms in the three DNA barcoding markers that allow rapid discrimination between Dalbergia from Madagascar and from other areas of its distribution range. Species identification success based on individual barcoding markers or combinations was poor, whereas subgroup identification success was much higher (up to 98%), revealing both the value and limitations of a DNA barcoding approach for the identification of closely related Malagasy rosewoods.

Integrating a comprehensive DNA barcode reference library with a global map of yews (Taxus L.) for forensic identification.

PubMed

Liu, Jie; Milne, Richard I; Möller, Michael; Zhu, Guang-Fu; Ye, Lin-Jiang; Luo, Ya-Huang; Yang, Jun-Bo; Wambulwa, Moses C; Wang, Chun-Neng; Li, De-Zhu; Gao, Lian-Ming

2018-05-22

Rapid and accurate identification of endangered species is a critical component of biosurveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or preprocessed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa. Here, we construct a comprehensive DNA barcode reference library and generate distribution maps using species distribution modelling (SDM), for all 15 Taxus species worldwide. We find that trnL-trnF is the ideal barcode for Taxus: It can distinguish all Taxus species and in combination with ITS identify hybrids. Among five analysis methods tested, NJ was the most effective. Among 4,151 individuals screened for trnL-trnF, 73 haplotypes were detected, all species-specific and some population private. Taxonomical, geographical and genetic dimensions of sampling strategy were all found to affect the comprehensiveness of the resulting DNA barcode library. Maps from SDM showed that most species had allopatric distributions, except T. mairei in the Sino-Himalayan region. Using the barcode library and distribution map data, two unknown forensic samples were identified to species (and in one case, population) level and another was determined as a putative interspecific hybrid. This integrated species identification system for Taxus can be used for biosurveillance, conservation management and to monitor and prosecute illegal trade. Similar identification systems are recommended for other IUCN- and CITES-listed taxa. © 2018 John Wiley & Sons Ltd.

Isolation and identification of female DNA on postcoital penile swabs.

PubMed

Cina, S J; Collins, K A; Pettenati, M J; Fitts, M

2000-06-01

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

PubMed

Normand, A C; Packeu, A; Cassagne, C; Hendrickx, M; Ranque, S; Piarroux, R

2018-05-01

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton , mainly T. rubrum complex ( n = 184), T. interdigitale ( n = 40), T. tonsurans ( n = 26), and T. benhamiae ( n = 5). Other genera included Microsporum (e.g., M. canis [ n = 21], M. audouinii [ n = 10], Nannizzia gypsea [ n = 3], and Epidermophyton [ n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified. Copyright © 2018 American Society for Microbiology.

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia.

PubMed

Osathanunkul, Maslin; Dheeranupattana, Srisulak; Rotarayanont, Siriphron; Sookkhee, Siriwoot; Osathanunkul, Khukrit; Madesis, Panagiotis

2017-12-02

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.

DNA barcoding and morphological identification of neotropical ichthyoplankton from the Upper Paraná and São Francisco.

PubMed

Becker, R A; Sales, N G; Santos, G M; Santos, G B; Carvalho, D C

2015-07-01

The identification of fish larvae from two neotropical hydrographic basins using traditional morphological taxonomy and DNA barcoding revealed no conflicting results between the morphological and barcode identification of larvae. A lower rate (25%) of correct morphological identification of eggs as belonging to migratory or non-migratory species was achieved. Accurate identification of ichthyoplankton by DNA barcoding is an important tool for fish reproductive behaviour studies, correct estimation of biodiversity by detecting eggs from rare species, as well as defining environmental and management strategies for fish conservation in the neotropics. © 2015 The Fisheries Society of the British Isles.

DNA Barcoding for Species Identification of Insect Skins: A Test on Chironomidae (Diptera) Pupal Exuviae

PubMed Central

Ekrem, Torbjørn; Stur, Elisabeth

2017-01-01

Abstract Chironomidae (Diptera) pupal exuviae samples are commonly used for biological monitoring of aquatic habitats. DNA barcoding has proved useful for species identification of chironomid life stages containing cellular tissue, but the barcoding success of chironomid pupal exuviae is unknown. We assessed whether standard DNA barcoding could be efficiently used for species identification of chironomid pupal exuviae when compared with morphological techniques and if there were differences in performance between temperate and tropical ecosystems, subfamilies, and tribes. PCR, sequence, and identification success differed significantly between geographic regions and taxonomic groups. For Norway, 27 out of 190 (14.2%) of pupal exuviae resulted in high-quality chironomid sequences that match species. For Costa Rica, 69 out of 190 (36.3%) Costa Rican pupal exuviae resulted in high-quality sequences, but none matched known species. Standard DNA barcoding of chironomid pupal exuviae had limited success in species identification of unknown specimens due to contaminations and lack of matching references in available barcode libraries, especially from Costa Rica. Therefore, we recommend future biodiversity studies that focus their efforts on understudied regions, to simultaneously use morphological and molecular identification techniques to identify all life stages of chironomids and populate the barcode reference library with identified sequences.

Identification of Scleractinian Coral Recruits Using Fluorescent Censusing and DNA Barcoding Techniques

PubMed Central

Hsu, Chia-Min; de Palmas, Stéphane; Kuo, Chao-Yang; Denis, Vianney; Chen, Chaolun Allen

2014-01-01

The identification of coral recruits has been problematic due to a lack of definitive morphological characters being available for higher taxonomic resolution. In this study, we tested whether fluorescent detection of coral recruits used in combinations of different DNA-barcoding markers (cytochrome oxidase I gene [COI], open reading frame [ORF], and nuclear Pax-C intron [PaxC]) could be useful for increasing the resolution of coral spat identification in ecological studies. One hundred and fifty settlement plates were emplaced at nine sites on the fringing reefs of Kenting National Park in southern Taiwan between April 2011 and September 2012. A total of 248 living coral spats and juveniles (with basal areas ranging from 0.21 to 134.57 mm2) were detected on the plates with the aid of fluorescent light and collected for molecular analyses. Using the COI DNA barcoding technique, 90.3% (224/248) of coral spats were successfully identified into six genera, including Acropora, Isopora, Montipora, Pocillopora, Porites, and Pavona. PaxC further separated I. cuneata and I. palifera of Isopora from Acropora, and ORF successfully identified the species of Pocillopora (except P. meandrina and P. eydouxi). Moreover, other cnidarian species such as actinarians, zoanthids, and Millepora species were visually found using fluorescence and identified by COI DNA barcoding. This combination of existing approaches greatly improved the taxonomic resolution of early coral life stages, which to date has been mainly limited to the family level based on skeletal identification. Overall, this study suggests important improvements for the identification of coral recruits in ecological studies. PMID:25211345

Identification of scleractinian coral recruits using fluorescent censusing and DNA barcoding techniques.

PubMed

Hsu, Chia-Min; de Palmas, Stéphane; Kuo, Chao-Yang; Denis, Vianney; Chen, Chaolun Allen

2014-01-01

The identification of coral recruits has been problematic due to a lack of definitive morphological characters being available for higher taxonomic resolution. In this study, we tested whether fluorescent detection of coral recruits used in combinations of different DNA-barcoding markers (cytochrome oxidase I gene [COI], open reading frame [ORF], and nuclear Pax-C intron [PaxC]) could be useful for increasing the resolution of coral spat identification in ecological studies. One hundred and fifty settlement plates were emplaced at nine sites on the fringing reefs of Kenting National Park in southern Taiwan between April 2011 and September 2012. A total of 248 living coral spats and juveniles (with basal areas ranging from 0.21 to 134.57 mm(2)) were detected on the plates with the aid of fluorescent light and collected for molecular analyses. Using the COI DNA barcoding technique, 90.3% (224/248) of coral spats were successfully identified into six genera, including Acropora, Isopora, Montipora, Pocillopora, Porites, and Pavona. PaxC further separated I. cuneata and I. palifera of Isopora from Acropora, and ORF successfully identified the species of Pocillopora (except P. meandrina and P. eydouxi). Moreover, other cnidarian species such as actinarians, zoanthids, and Millepora species were visually found using fluorescence and identified by COI DNA barcoding. This combination of existing approaches greatly improved the taxonomic resolution of early coral life stages, which to date has been mainly limited to the family level based on skeletal identification. Overall, this study suggests important improvements for the identification of coral recruits in ecological studies.

The influence of diet on faecal DNA amplification and sex identification in brown bears (Ursus arctos)

USGS Publications Warehouse

Murphy, M.A.; Waits, L.P.; Kendall, K.C.

2003-01-01

To evaluate the influence of diet on faecal DNA amplification, 11 captive brown bears (Ursus arctos) were placed on six restricted diets: grass (Trifolium spp., Haplopappus hirtus and Poa pratensis), alfalfa (Lupinus spp.), carrots (Daucus spp.), white-tailed deer (Odocoileus virginianus), blueberries (Vaccinium spp.) and salmon (Salmo spp.). DNA was extracted from 50 faecal samples of each restricted diet, and amplification of brown bear DNA was attempted for a mitochondrial DNA (mtDNA) locus and nuclear DNA (nDNA) locus. For mtDNA, no significant differences were observed in amplification success rates across diets. For nDNA, amplification success rates for salmon diet extracts were significantly lower than all other diet extracts (P < 0.001). To evaluate the accuracy of faecal DNA sex identification when female carnivores consume male mammalian prey, female bears were fed male white-tailed deer. Four of 10 extracts amplified, and all extracts were incorrectly scored as male due to amplification of X and Y-chromosome fragments. The potential biases highlighted in this study have broad implications for researchers using faecal DNA for individual and sex identification, and should be evaluated in other species.

DNA-based identification and phylogeny of North American Armillaria species

Treesearch

Amy L. Ross-Davis; John W. Hanna; Ned B. Klopfenstein

2011-01-01

Because Armillaria species display different ecological behaviors across diverse forest ecosystems, it is critical to identify Armillaria species accurately for any assessment of forest health. To further develop DNA-based identification methods, partial sequences of the translation elongation factor-1 alpha (EF-1α) gene were used to examine the phylogenetic...

Avatar DNA Nanohybrid System in Chip-on-a-Phone

NASA Astrophysics Data System (ADS)

Park, Dae-Hwan; Han, Chang Jo; Shul, Yong-Gun; Choy, Jin-Ho

2014-05-01

Long admired for informational role and recognition function in multidisciplinary science, DNA nanohybrids have been emerging as ideal materials for molecular nanotechnology and genetic information code. Here, we designed an optical machine-readable DNA icon on microarray, Avatar DNA, for automatic identification and data capture such as Quick Response and ColorZip codes. Avatar icon is made of telepathic DNA-DNA hybrids inscribed on chips, which can be identified by camera of smartphone with application software. Information encoded in base-sequences can be accessed by connecting an off-line icon to an on-line web-server network to provide message, index, or URL from database library. Avatar DNA is then converged with nano-bio-info-cogno science: each building block stands for inorganic nanosheets, nucleotides, digits, and pixels. This convergence could address item-level identification that strengthens supply-chain security for drug counterfeits. It can, therefore, provide molecular-level vision through mobile network to coordinate and integrate data management channels for visual detection and recording.

Avatar DNA Nanohybrid System in Chip-on-a-Phone

PubMed Central

Park, Dae-Hwan; Han, Chang Jo; Shul, Yong-Gun; Choy, Jin-Ho

2014-01-01

Long admired for informational role and recognition function in multidisciplinary science, DNA nanohybrids have been emerging as ideal materials for molecular nanotechnology and genetic information code. Here, we designed an optical machine-readable DNA icon on microarray, Avatar DNA, for automatic identification and data capture such as Quick Response and ColorZip codes. Avatar icon is made of telepathic DNA-DNA hybrids inscribed on chips, which can be identified by camera of smartphone with application software. Information encoded in base-sequences can be accessed by connecting an off-line icon to an on-line web-server network to provide message, index, or URL from database library. Avatar DNA is then converged with nano-bio-info-cogno science: each building block stands for inorganic nanosheets, nucleotides, digits, and pixels. This convergence could address item-level identification that strengthens supply-chain security for drug counterfeits. It can, therefore, provide molecular-level vision through mobile network to coordinate and integrate data management channels for visual detection and recording. PMID:24824876

Patient identification error among prostate needle core biopsy specimens--are we ready for a DNA time-out?

PubMed

Suba, Eric J; Pfeifer, John D; Raab, Stephen S

2007-10-01

Patient identification errors in surgical pathology often involve switches of prostate or breast needle core biopsy specimens among patients. We assessed strategies for decreasing the occurrence of these uncommon and yet potentially catastrophic events. Root cause analyses were performed following 3 cases of patient identification error involving prostate needle core biopsy specimens. Patient identification errors in surgical pathology result from slips and lapses of automatic human action that may occur at numerous steps during pre-laboratory, laboratory and post-laboratory work flow processes. Patient identification errors among prostate needle biopsies may be difficult to entirely prevent through the optimization of work flow processes. A DNA time-out, whereby DNA polymorphic microsatellite analysis is used to confirm patient identification before radiation therapy or radical surgery, may eliminate patient identification errors among needle biopsies.

DMINDA: an integrated web server for DNA motif identification and analyses

PubMed Central

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-01-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. PMID:24753419

DNA analysis in perpetrator identification of terrorism-related disaster: suicide bombing of the Australian Embassy in Jakarta 2004.

PubMed

Sudoyo, Herawati; Widodo, Putut T; Suryadi, Helena; Lie, Yuliana S; Safari, Dodi; Widjajanto, Agung; Kadarmo, D Aji; Hidayat, Soegeng; Marzuki, Sangkot

2008-06-01

We report the strategy that we employed to identify the perpetrator of a suicide car bombing in front of the Australian Embassy in Jakarta, Indonesia, on 9 September 2004. The bomb was so massive that only small tissue pieces of the perpetrator could be recovered, preventing conventional approach to the identification of the bomber, necessitating the introduction of DNA analysis as the primary means for perpetrator identification. Crime scene investigation revealed the trajectory of the bomb blast, which was used to guide the collection of charred tissue fragments of the perpetrator. Mitochondrial DNA analysis was first conducted on 17 tissue fragments, recovered over large areas of the trajectory to, (a) confirm that they are of a common source, i.e. the perpetrator, and thus (b) establish the mtDNA HV1 sequence profile of the perpetrator. The mtDNA of the perpetrator matches that of a maternally related family member of one of four suspects. Standard autosomal STR analysis confirmed the identification. This case is of interest as an illustration of a successful application of DNA analysis as the primary means of disaster perpetrator identification.

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

PubMed

Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

2015-11-01

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

Application of permanents of square matrices for DNA identification in multiple-fatality cases

PubMed Central

2013-01-01

Background DNA profiling is essential for individual identification. In forensic medicine, the likelihood ratio (LR) is commonly used to identify individuals. The LR is calculated by comparing two hypotheses for the sample DNA: that the sample DNA is identical or related to a reference DNA, and that it is randomly sampled from a population. For multiple-fatality cases, however, identification should be considered as an assignment problem, and a particular sample and reference pair should therefore be compared with other possibilities conditional on the entire dataset. Results We developed a new method to compute the probability via permanents of square matrices of nonnegative entries. As the exact permanent is known as a #P-complete problem, we applied the Huber–Law algorithm to approximate the permanents. We performed a computer simulation to evaluate the performance of our method via receiver operating characteristic curve analysis compared with LR under the assumption of a closed incident. Differences between the two methods were well demonstrated when references provided neither obligate alleles nor impossible alleles. The new method exhibited higher sensitivity (0.188 vs. 0.055) at a threshold value of 0.999, at which specificity was 1, and it exhibited higher area under a receiver operating characteristic curve (0.990 vs. 0.959, P = 9.6E-15). Conclusions Our method therefore offers a solution for a computationally intensive assignment problem and may be a viable alternative to LR-based identification for closed-incident multiple-fatality cases. PMID:23962363

Identification of Neoceratitis asiatica (Becker) (Diptera: Tephritidae) based on morphological characteristics and DNA barcode.

PubMed

Guo, Shaokun; He, Jia; Zhao, Zihua; Liu, Lijun; Gao, Liyuan; Wei, Shuhua; Guo, Xiaoyu; Zhang, Rong; Li, Zhihong

2017-12-12

Neoceratitis asiatica (Becker), which especially infests wolfberry (Lycium barbarum L.), could cause serious economic losses every year in China, especially to organic wolfberry production. In some important wolfberry plantings, it is difficult and time-consuming to rear the larvae or pupae to adults for morphological identification. Molecular identification based on DNA barcode is a solution to the problem. In this study, 15 samples were collected from Ningxia, China. Among them, five adults were identified according to their morphological characteristics. The utility of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) gene sequence as DNA barcode in distinguishing N. asiatica was evaluated by analysing Kimura 2-parameter distances and phylogenetic trees. There were significant differences between intra-specific and inter-specific genetic distances according to the barcoding gap analysis. The uncertain larval and pupal samples were within the same cluster as N. asiatica adults and formed sister cluster to N. cyanescens. A combination of morphological and molecular methods enabled accurate identification of N. asiatica. This is the first study using DNA barcode to identify N. asiatica and the obtained DNA sequences will be added to the DNA barcode database.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

PubMed

Najafzadeh, M J; Vicente, V A; Feng, Peiying; Naseri, A; Sun, Jiufeng; Rezaei-Matehkolaei, A; de Hoog, G S

2018-03-05

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

DNA-Based Identification of Forensically Important Blow Flies (Diptera: Calliphoridae) From India.

PubMed

Bharti, Meenakshi; Singh, Baneshwar

2017-09-01

Correct species identification is the first and the most important criteria in entomological evidence-based postmortem interval (PMI) estimation. Although morphological keys are available for species identification of adult blow flies, keys for immature stages are either lacking or are incomplete. In this study, cytochrome oxidase subunit 1 (COI) reference data were developed from nine species (belonging to three subfamilies, namely, Calliphorinae, Luciliinae, and Chrysomyinae) of blow flies from India. Seven of the nine species included in this study were found suitable for DNA-based identification using COI gene, because they showed nonoverlapping intra- (0.0-0.3%) and inter-(1.96-18.14%) specific diversity, and formed well-supported monophyletic clade in phylogenetic analysis. The remaining two species (i.e., Chrysomya megacephala (Fabricius) and Chrysomya chani Kurahashi) cannot be distinguished reliably using our database because they had a very low interspecific diversity (0.11%), and Ch. megacephala was paraphyletic with respect to Ch. chani in the phylogenetic analysis. We conclude that the COI gene is a useful marker for DNA-based identification of blow flies from India. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

NASA Astrophysics Data System (ADS)

Millard, Julie T.; Pilon, André M.

2003-04-01

A recent forensic approach for identification of unknown biological samples is mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify a 440 base pair hypervariable region of human mtDNA from a variety of "crime scene" samples (e.g., teeth, hair, nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence alignments are made via the program CLUSTAL W, allowing students to compare samples and solve the "crime."

Development of a New Marker System for Identification of Spirodela polyrhiza and Landoltia punctata

PubMed Central

Feng, Bo; Fang, Yang; Xu, Zhibin; Xiang, Chao; Zhou, Chunhong; Jiang, Fei; Wang, Tao

2017-01-01

Lemnaceae (commonly called duckweed) is an aquatic plant ideal for quantitative analysis in plant sciences. Several species of this family represent the smallest and fastest growing flowering plants. Different ecotypes of the same species vary in their biochemical and physiological properties. Thus, selecting of desirable ecotypes of a species is very important. Here, we developed a simple and rapid molecular identification system for Spirodela polyrhiza and Landoltia punctata based on the sequence polymorphism. First, several pairs of primers were designed and three markers were selected as good for identification. After PCR amplification, DNA fragments (the combination of three PCR products) in different duckweeds were detected using capillary electrophoresis. The high-resolution capillary electrophoresis displayed high identity to the sequencing results. The combination of the PCR products containing several DNA fragments highly improved the identification frequency. These results indicate that this method is not only good for interspecies identification but also ideal for intraspecies distinguishing. Meanwhile, 11 haplotypes were found in both the S. polyrhiza and L. punctata ecotypes. The results suggest that this marker system is useful for large-scale identification of duckweed and for the screening of desirable ecotypes to improve the diverse usage in duckweed utilization. PMID:28168191

Forensic identification of Indian snakeroot (Rauvolfia serpentina Benth. ex Kurz) using DNA barcoding.

PubMed

Eurlings, Marcel C M; Lens, Frederic; Pakusza, Csilla; Peelen, Tamara; Wieringa, Jan J; Gravendeel, Barbara

2013-05-01

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species-specific DNA polymorphisms. We found two species-specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species-specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot. © 2013 American Academy of Forensic Sciences.

DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

2017-02-01

Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

Identification of propulsion systems

NASA Technical Reports Server (NTRS)

Merrill, Walter; Guo, Ten-Huei; Duyar, Ahmet

1991-01-01

This paper presents a tutorial on the use of model identification techniques for the identification of propulsion system models. These models are important for control design, simulation, parameter estimation, and fault detection. Propulsion system identification is defined in the context of the classical description of identification as a four step process that is unique because of special considerations of data and error sources. Propulsion system models are described along with the dependence of system operation on the environment. Propulsion system simulation approaches are discussed as well as approaches to propulsion system identification with examples for both air breathing and rocket systems.

[A study of culture-based easy identification system for Malassezia].

PubMed

Kaneko, Takamasa

2011-01-01

Most species of this genus are lipid-dependent yeasts, which colonize the seborrheic part of the skin, and they have been reported to be associated with pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Malassezia have been re-classified into 7 species based on molecular biological analysis of nuclear ribosomal DNA/RNA and new Malassezia species were reported. As members of the genus Malassezia share similar morphological and biochemical characteristics, it was thought to be difficult to differentiate between them based on phenotypic features. While molecular biological techniques are the most reliable methods for identification of Malassezia, they are not available in most clinical laboratories. We studied ( i ) development of an efficient isolation media and culture based easy identification system, ( ii ) the incidence of atypical biochemical features in Malassezia species and propose a culture-based easy identification system for clinically important Malassezia species, M. globosa, M. restricta, and M. furfur.

DMINDA: an integrated web server for DNA motif identification and analyses.

PubMed

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-07-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of food and beverage spoilage yeasts from DNA sequence analyses

USDA-ARS?s Scientific Manuscript database

Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

Mass spectrometry-based cDNA profiling as a potential tool for human body fluid identification.

PubMed

Donfack, Joseph; Wiley, Anissa

2015-05-01

Several mRNA markers have been exhaustively evaluated for the identification of human venous blood, saliva, and semen in forensic genetics. As new candidate human body fluid specific markers are discovered, evaluated, and reported in the scientific literature, there is an increasing trend toward determining the ideal markers for cDNA profiling of body fluids of forensic interest. However, it has not been determined which molecular genetics-based technique(s) should be utilized to assess the performance of these markers. In recent years, only a few confirmatory, mRNA/cDNA-based methods have been evaluated for applications in body fluid identification. The most frequently described methods tested to date include quantitative polymerase chain reaction (qPCR) and capillary electrophoresis (CE). However these methods, in particular qPCR, often favor narrow multiplex PCR due to the availability of a limited number of fluorescent dyes/tags. In an attempt to address this technological constraint, this study explored matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for human body fluid identification via cDNA profiling of venous blood, saliva, and semen. Using cDNA samples at 20pg input phosphoglycerate kinase 1 (PGK1) amounts, body fluid specific markers for the candidate genes were amplified in their corresponding body fluid (i.e., venous blood, saliva, or semen) and absent in the remaining two (100% specificity). The results of this study provide an initial indication that MALDI-TOF MS is a potential fluorescent dye-free alternative method for body fluid identification in forensic casework. However, the inherent issues of low amounts of mRNA, and the damage caused to mRNA by environmental exposures, extraction processes, and storage conditions are important factors that significantly hinder the implementation of cDNA profiling into forensic casework. Published by Elsevier Ireland Ltd.

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions

PubMed Central

Raupach, Michael J.; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C.; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences. PMID:26417993

The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions.

PubMed

Raupach, Michael J; Barco, Andrea; Steinke, Dirk; Beermann, Jan; Laakmann, Silke; Mohrbeck, Inga; Neumann, Hermann; Kihara, Terue C; Pointner, Karin; Radulovici, Adriana; Segelken-Voigt, Alexandra; Wesse, Christina; Knebelsberger, Thomas

2015-01-01

During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD), unique BINs were identified for 198 (96.6%) of the analyzed species. Six species were characterized by two BINs (2.9%), and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%). Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%). Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761), underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

[DNA prints instead of plantar prints in neonatal identification].

PubMed

Rodríguez-Alarcón Gómez, J; Martińez de Pancorbo Gómez, M; Santillana Ferrer, L; Castro Espido, A; Melchor Maros, J C; Linares Uribe, M A; Fernández-Llebrez del Rey, L; Aranguren Dúo, G

1996-06-22

To check the possible usefulness in studying DNA in dried blood spots taken on filter paper blotters for newborn identification. It set out to establish: 1. The validity of the method for analysis; 2. The validity of all stored samples (such as those kept in clinical records); 3. Guarantee of non-intrusion in the genetic code; 4. Acceptable price and execution time. Forty (40) anonymous 13-year-old samples of 20 subjects (2 per subject) were studied. DNA was extracted using Chelex resin and the STR ("small tandem repeat") of microsatellite DNA was studies using the "polimerase chain reaction method" (PCR). Three non coding DNA loci (CSF1PO, TPOX and THO1) were analyzed by Multiplex amplification. It was possible to type 39 samples, making it possible to match the 20 cases (one by exclusion). The complete procedure yielded the results within 24 hours in all cases. The estimated final cost was found to be a fifth of that conventional maternity/paternity tests. The study carried out made matching possible in all 20 cases (directly in 19 cases). It was not necessary to study DNA coding areas. The validity of the method for analyzing samples stored for 13 years without any special care was also demonstrated. The technic was fast, producing the results within 24 hours, and at reasonable cost.

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

PubMed Central

Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta; Kawube, Geofrey; Bertaccini, Assunta; Nicolaisen, Mogens

2012-01-01

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. PMID:23272216

DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina.

PubMed

Čakar, Jasmina; Pilav, Amela; Džehverović, Mirela; Ahatović, Anesa; Haverić, Sanin; Ramić, Jasmin; Marjanović, Damir

2018-01-01

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of Šerići, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex ® Fusion and PowerPlex ® Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains. © 2017 American Academy of Forensic Sciences.

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

PubMed

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

NASA Astrophysics Data System (ADS)

Mulvaney, Shawn P.; Ibe, Carol N.; Caldwell, Jane M.; Levine, Jay F.; Whitman, Lloyd J.; Tamanaha, Cy R.

2009-02-01

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

Correlation dynamics and enhanced signals for the identification of serial biomolecules and DNA bases.

PubMed

Ahmed, Towfiq; Haraldsen, Jason T; Rehr, John J; Di Ventra, Massimiliano; Schuller, Ivan; Balatsky, Alexander V

2014-03-28

Nanopore-based sequencing has demonstrated a significant potential for the development of fast, accurate, and cost-efficient fingerprinting techniques for next generation molecular detection and sequencing. We propose a specific multilayered graphene-based nanopore device architecture for the recognition of single biomolecules. Molecular detection and analysis can be accomplished through the detection of transverse currents as the molecule or DNA base translocates through the nanopore. To increase the overall signal-to-noise ratio and the accuracy, we implement a new 'multi-point cross-correlation' technique for identification of DNA bases or other molecules on the single molecular level. We demonstrate that the cross-correlations between each nanopore will greatly enhance the transverse current signal for each molecule. We implement first-principles transport calculations for DNA bases surveyed across a multilayered graphene nanopore system to illustrate the advantages of the proposed geometry. A time-series analysis of the cross-correlation functions illustrates the potential of this method for enhancing the signal-to-noise ratio. This work constitutes a significant step forward in facilitating fingerprinting of single biomolecules using solid state technology.

Development of a DNA microarray for species identification of quarantine aphids.

PubMed

Lee, Won Sun; Choi, Hwalran; Kang, Jinseok; Kim, Ji-Hoon; Lee, Si Hyeock; Lee, Seunghwan; Hwang, Seung Yong

2013-12-01

Aphid pests are being brought into Korea as a result of increased crop trading. Aphids exist on growth areas of plants, and thus plant growth is seriously affected by aphid pests. However, aphids are very small and have several sexual morphs and life stages, so it is difficult to identify species on the basis of morphological features. This problem was approached using DNA microarray technology. DNA targets of the cytochrome c oxidase subunit I gene were generated with a fluorescent dye-labelled primer and were hybridised onto a DNA microarray consisting of specific probes. After analysing the signal intensity of the specific probes, the unique patterns from the DNA microarray, consisting of 47 species-specific probes, were obtained to identify 23 aphid species. To confirm the accuracy of the developed DNA microarray, ten individual blind samples were used in blind trials, and the identifications were completely consistent with the sequencing data of all individual blind samples. A microarray has been developed to distinguish aphid species. DNA microarray technology provides a rapid, easy, cost-effective and accurate method for identifying aphid species for pest control management. © 2013 Society of Chemical Industry.

Benefits and challenges to using DNA-based identification methods: An example study of larval fish from nearshore areas of Lake Superior

EPA Science Inventory

DNA-based identification methods could increase the ability of aquatic resource managers to track patterns of invasive species, especially for taxa that are difficult to identify morphologically. Nonetheless, use of DNA-based identification methods in aquatic surveys is still unc...

Real-Time Continuous Identification of Greenhouse Plant Pathogens Based on Recyclable Microfluidic Bioassay System.

PubMed

Qu, Xiangmeng; Li, Min; Zhang, Hongbo; Lin, Chenglie; Wang, Fei; Xiao, Mingshu; Zhou, Yi; Shi, Jiye; Aldalbahi, Ali; Pei, Hao; Chen, Hong; Li, Li

2017-09-20

The development of a real-time continuous analytical platform for the pathogen detection is of great scientific importance for achieving better disease control and prevention. In this work, we report a rapid and recyclable microfluidic bioassay system constructed from oligonucleotide arrays for selective and sensitive continuous identification of DNA targets of fungal pathogens. We employ the thermal denaturation method to effectively regenerate the oligonucleotide arrays for multiple sample detection, which could considerably reduce the screening effort and costs. The combination of thermal denaturation and laser-induced fluorescence detection technique enables real-time continuous identification of multiple samples (<10 min per sample). As a proof of concept, we have demonstrated that two DNA targets of fungal pathogens (Botrytis cinerea and Didymella bryoniae) can be sequentially analyzed using our rapid microfluidic bioassay system, which provides a new paradigm in the design of microfluidic bioassay system and will be valuable for chemical and biomedical analysis.

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences.

PubMed

Chung, In-Hyuk; Yoo, Hye Sook; Eah, Jae-Yong; Yoon, Hyun-Kyu; Jung, Jin-Wook; Hwang, Seung Yong; Kim, Chang-Bae

2010-10-01

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

PubMed

Yang, G; Ding, J; Wu, L R; Duan, Y D; Li, A Y; Shan, J Y; Wu, Y X

2015-03-13

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

Issues and strategies in the DNA identification of World Trade Center victims.

PubMed

Brenner, C H; Weir, B S

2003-05-01

Identification of the nearly 3000 victims of the World Trade Center attack, represented by about 15,000 body parts, rests heavily on DNA. Reference DNA profiles are often from relatives rather than from the deceased themselves. With so large a set of victims, coincidental similarities between non-relatives abound. Therefore considerable care is necessary to succeed in correlating references with correct victims while avoiding spurious assignments. Typically multiple relatives are necessary to establish the identity of a victim. We describe a 3-stage paradigm--collapse, screen, test--to organize the work of sorting out the identities. Inter alia we present a simple and general formula for the likelihood ratio governing practically any potential relationship between two DNA profiles.

Forensic DNA Profiling and Database

PubMed Central

Panneerchelvam, S.; Norazmi, M.N.

2003-01-01

The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

DEVELOPMENT OF DNA-BASED TOOLS FOR IDENTIFICATION AND MONITORING OF AQUATIC INTRODUCED SPECIES

EPA Science Inventory

Claims for potential applications of DNA taxonomy range from identification of unknown specimens and the discovery of new species to the study of biodiversity through comprehensive characterizations of complex biotic communities drawn from environmental samples. Recently, these a...

Inclusion probability for DNA mixtures is a subjective one-sided match statistic unrelated to identification information.

PubMed

Perlin, Mark William

2015-01-01

DNA mixtures of two or more people are a common type of forensic crime scene evidence. A match statistic that connects the evidence to a criminal defendant is usually needed for court. Jurors rely on this strength of match to help decide guilt or innocence. However, the reliability of unsophisticated match statistics for DNA mixtures has been questioned. The most prevalent match statistic for DNA mixtures is the combined probability of inclusion (CPI), used by crime labs for over 15 years. When testing 13 short tandem repeat (STR) genetic loci, the CPI(-1) value is typically around a million, regardless of DNA mixture composition. However, actual identification information, as measured by a likelihood ratio (LR), spans a much broader range. This study examined probability of inclusion (PI) mixture statistics for 517 locus experiments drawn from 16 reported cases and compared them with LR locus information calculated independently on the same data. The log(PI(-1)) values were examined and compared with corresponding log(LR) values. The LR and CPI methods were compared in case examples of false inclusion, false exclusion, a homicide, and criminal justice outcomes. Statistical analysis of crime laboratory STR data shows that inclusion match statistics exhibit a truncated normal distribution having zero center, with little correlation to actual identification information. By the law of large numbers (LLN), CPI(-1) increases with the number of tested genetic loci, regardless of DNA mixture composition or match information. These statistical findings explain why CPI is relatively constant, with implications for DNA policy, criminal justice, cost of crime, and crime prevention. Forensic crime laboratories have generated CPI statistics on hundreds of thousands of DNA mixture evidence items. However, this commonly used match statistic behaves like a random generator of inclusionary values, following the LLN rather than measuring identification information. A quantitative

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

PubMed

Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

2006-05-01

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains.

PubMed

Royle, Thomas C A; Sakhrani, Dionne; Speller, Camilla F; Butler, Virginia L; Devlin, Robert H; Cannon, Aubrey; Yang, Dongya Y

2018-01-01

Pacific salmonid (Oncorhynchus spp.) remains are routinely recovered from archaeological sites in northwestern North America but typically lack sexually dimorphic features, precluding the sex identification of these remains through morphological approaches. Consequently, little is known about the deep history of the sex-selective salmonid fishing strategies practiced by some of the region's Indigenous peoples. Here, we present a DNA-based method for the sex identification of archaeological Pacific salmonid remains that integrates two PCR assays that each co-amplify fragments of the sexually dimorphic on the Y chromosome (sdY) gene and an internal positive control (Clock1a or D-loop). The first assay co-amplifies a 95 bp fragment of sdY and a 108 bp fragment of the autosomal Clock1a gene, whereas the second assay co-amplifies the same sdY fragment and a 249 bp fragment of the mitochondrial D-loop region. This method's reliability, sensitivity, and efficiency, were evaluated by applying it to 72 modern Pacific salmonids from five species and 75 archaeological remains from six Pacific salmonids. The sex identities assigned to each of the modern samples were concordant with their known phenotypic sex, highlighting the method's reliability. Applications of the method to dilutions of modern DNA samples indicate it can correctly identify the sex of samples with as little as ~39 pg of total genomic DNA. The successful sex identification of 70 of the 75 (93%) archaeological samples further demonstrates the method's sensitivity. The method's reliance on two co-amplifications that preferentially amplify sdY helps validate the sex identities assigned to samples and reduce erroneous identifications caused by allelic dropout and contamination. Furthermore, by sequencing the D-loop fragment used as a positive control, species-level and sex identifications can be simultaneously assigned to samples. Overall, our results indicate the DNA-based method reported in this study is a

Identification of species based on DNA barcode using k-mer feature vector and Random forest classifier.

PubMed

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R

2016-11-05

DNA barcoding is a molecular diagnostic method that allows automated and accurate identification of species based on a short and standardized fragment of DNA. To this end, an attempt has been made in this study to develop a computational approach for identifying the species by comparing its barcode with the barcode sequence of known species present in the reference library. Each barcode sequence was first mapped onto a numeric feature vector based on k-mer frequencies and then Random forest methodology was employed on the transformed dataset for species identification. The proposed approach outperformed similarity-based, tree-based, diagnostic-based approaches and found comparable with existing supervised learning based approaches in terms of species identification success rate, while compared using real and simulated datasets. Based on the proposed approach, an online web interface SPIDBAR has also been developed and made freely available at http://cabgrid.res.in:8080/spidbar/ for species identification by the taxonomists. Copyright © 2016 Elsevier B.V. All rights reserved.

FBIS: A regional DNA barcode archival & analysis system for Indian fishes.

PubMed

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. The database is available for free at http://mail.nbfgr.res.in/fbis/

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

PubMed Central

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

Identification of unique repeated patterns, location of mutation in DNA finger printing using artificial intelligence technique.

PubMed

Mukunthan, B; Nagaveni, N

2014-01-01

In genetic engineering, conventional techniques and algorithms employed by forensic scientists to assist in identification of individuals on the basis of their respective DNA profiles involves more complex computational steps and mathematical formulae, also the identification of location of mutation in a genomic sequence in laboratories is still an exigent task. This novel approach provides ability to solve the problems that do not have an algorithmic solution and the available solutions are also too complex to be found. The perfect blend made of bioinformatics and neural networks technique results in efficient DNA pattern analysis algorithm with utmost prediction accuracy.

Prospects and Problems for Identification of Poisonous Plants in China using DNA Barcodes.

PubMed

Xie, Lei; Wang, Ying Wei; Guan, Shan Yue; Xie, Li Jing; Long, Xin; Sun, Cheng Ye

2014-10-01

Poisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China. Seventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes (matK, rbcL, and ITS) were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment (PWG) distance, and Tree-Building were tested for discrimination power. The primer universality of all the three markers was high. Except in the case of ITS for Hemerocallis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method. DNA barcoding technique provides us a fast tool for clinical identification of poisonous plants in China. We suggest matK, rbcL, ITS used in combination as DNA barcodes for authentication of poisonous plants. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

PubMed

Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

2017-06-21

The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing

DNA barcode identification of commercial fish sold in Mexican markets.

PubMed

Sarmiento-Camacho, Stephanie; Valdez-Moreno, Martha

2018-04-24

The substitution of high-value fish species for those of lower value is common practice. Although numerous studies have addressed this issue, few have been conducted in Mexico. In this study, we sought to identify fresh fillets of fish, sharks, and rays using DNA barcodes. We analyzed material from "La Viga" in Mexico City, and other markets located on the Gulf and Caribbean coasts of Mexico. From 134 samples, we obtained sequences from 129, identified to 9 orders, 28 families, 38 genera, and 44 species. The most common species were Seriola dumerili, Pangasianodon hypophthalmus, Carcharhinus falciformis, Carcharhinus brevipinna, and Hypanus americanus. Pangasianodon hypophthalmus was most commonly used as a substitute for higher-value species. The substitution rate was 18% of the total. A review of the conservation status of the specimens identified against the IUNC list enabled us to establish that some species marketed in Mexico are threatened: Makaira nigricans, Lachnolaimus maximus, Hyporthodus flavolimbatus, and Isurus oxyrinchus are classified as vulnerable; Lopholatilus chamaeleonticeps and Sphyrna lewini are endangered; and the status of Hyporthodus nigritus is critical. These results will demonstrate to the Mexican authorities that DNA barcoding is a reliable tool for species identification, even when morphological identification is difficult or impossible.

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

A Next Generation Semiconductor Based Sequencing Approach for the Identification of Meat Species in DNA Mixtures

PubMed Central

Bertolini, Francesca; Ghionda, Marco Ciro; D’Alessandro, Enrico; Geraci, Claudia; Chiofalo, Vincenzo; Fontanesi, Luca

2015-01-01

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures. PMID:25923709

A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures.

PubMed

Bertolini, Francesca; Ghionda, Marco Ciro; D'Alessandro, Enrico; Geraci, Claudia; Chiofalo, Vincenzo; Fontanesi, Luca

2015-01-01

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

Identification of processed Chinese medicinal materials using DNA mini-barcoding.

PubMed

Song, Ming; Dong, Gang-Qiang; Zhang, Ya-Qin; Liu, Xia; Sun, Wei

2017-07-01

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psbA-trnH, rbcL, matK, trnL (UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL (UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%-20% of the processed samples, while the amplification rates of the trnL (UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL (UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

PubMed

Waleron, K; Waleron, M; Osipiuk, J; Podhajska, A J; Lojkowska, E

2006-02-01

Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

PubMed Central

Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

2013-01-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

PubMed

Iyer, Lakshminarayan M; Zhang, Dapeng; Burroughs, A Maxwell; Aravind, L

2013-09-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and

FaSTR DNA: a new expert system for forensic DNA analysis.

PubMed

Power, Timothy; McCabe, Brendan; Harbison, Sally Ann

2008-06-01

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i(3) v4 (The Forensic Science Service((R)) DNA expert System Suite FSS-i(3), Forensic Science Service, Birmingham, UK) with GeneScan Analysis v3.7/Genotyper v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper ID v3.2 and superior to that of FSS-i(3) v4 for the analysis of DNA profiles from crime samples.

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

PubMed

Hernández-Triana, Luis M; Montes De Oca, Fernanda; Prosser, Sean W J; Hebert, Paul D N; Gregory, T Ryan; McMurtrie, Shelley

2017-04-01

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%-4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance.

PubMed

Hsieh, Ying-Hsin; Wang, Yun F; Moura, Hercules; Miranda, Nancy; Simpson, Steven; Gowrishankar, Ramnath; Barr, John; Kerdahi, Khalil; Sulaiman, Irshad M

2018-05-01

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

FragIdent--automatic identification and characterisation of cDNA-fragments.

PubMed

Seelow, Dominik; Goehler, Heike; Hoffmann, Katrin

2009-03-02

Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments. Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available. Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones. We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at http://compbio.charite.de/genetik/FragIdent/.

Optimized System Identification

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan; Longman, Richard W.

1999-01-01

In system identification, one usually cares most about finding a model whose outputs are as close as possible to the true system outputs when the same input is applied to both. However, most system identification algorithms do not minimize this output error. Often they minimize model equation error instead, as in typical least-squares fits using a finite-difference model, and it is seen here that this distinction is significant. Here, we develop a set of system identification algorithms that minimize output error for multi-input/multi-output and multi-input/single-output systems. This is done with sequential quadratic programming iterations on the nonlinear least-squares problems, with an eigendecomposition to handle indefinite second partials. This optimization minimizes a nonlinear function of many variables, and hence can converge to local minima. To handle this problem, we start the iterations from the OKID (Observer/Kalman Identification) algorithm result. Not only has OKID proved very effective in practice, it minimizes an output error of an observer which has the property that as the data set gets large, it converges to minimizing the criterion of interest here. Hence, it is a particularly good starting point for the nonlinear iterations here. Examples show that the methods developed here eliminate the bias that is often observed using any system identification methods of either over-estimating or under-estimating the damping of vibration modes in lightly damped structures.

Inclusion probability for DNA mixtures is a subjective one-sided match statistic unrelated to identification information

PubMed Central

Perlin, Mark William

2015-01-01

Background: DNA mixtures of two or more people are a common type of forensic crime scene evidence. A match statistic that connects the evidence to a criminal defendant is usually needed for court. Jurors rely on this strength of match to help decide guilt or innocence. However, the reliability of unsophisticated match statistics for DNA mixtures has been questioned. Materials and Methods: The most prevalent match statistic for DNA mixtures is the combined probability of inclusion (CPI), used by crime labs for over 15 years. When testing 13 short tandem repeat (STR) genetic loci, the CPI-1 value is typically around a million, regardless of DNA mixture composition. However, actual identification information, as measured by a likelihood ratio (LR), spans a much broader range. This study examined probability of inclusion (PI) mixture statistics for 517 locus experiments drawn from 16 reported cases and compared them with LR locus information calculated independently on the same data. The log(PI-1) values were examined and compared with corresponding log(LR) values. Results: The LR and CPI methods were compared in case examples of false inclusion, false exclusion, a homicide, and criminal justice outcomes. Statistical analysis of crime laboratory STR data shows that inclusion match statistics exhibit a truncated normal distribution having zero center, with little correlation to actual identification information. By the law of large numbers (LLN), CPI-1 increases with the number of tested genetic loci, regardless of DNA mixture composition or match information. These statistical findings explain why CPI is relatively constant, with implications for DNA policy, criminal justice, cost of crime, and crime prevention. Conclusions: Forensic crime laboratories have generated CPI statistics on hundreds of thousands of DNA mixture evidence items. However, this commonly used match statistic behaves like a random generator of inclusionary values, following the LLN rather than

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

PubMed

de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

The effect on cadaver blood DNA identification by the use of targeted and whole body post-mortem computed tomography angiography.

PubMed

Rutty, Guy N; Barber, Jade; Amoroso, Jasmin; Morgan, Bruno; Graham, Eleanor A M

2013-12-01

Post-mortem computed tomography angiography (PMCTA) involves the injection of contrast agents. This could have both a dilution effect on biological fluid samples and could affect subsequent post-contrast analytical laboratory processes. We undertook a small sample study of 10 targeted and 10 whole body PMCTA cases to consider whether or not these two methods of PMCTA could affect post-PMCTA cadaver blood based DNA identification. We used standard methodology to examine DNA from blood samples obtained before and after the PMCTA procedure. We illustrate that neither of these PMCTA methods had an effect on the alleles called following short tandem repeat based DNA profiling, and therefore the ability to undertake post-PMCTA blood based DNA identification.

Identification and characterization of a DnaJ gene from red alga Pyropia yezoensis (Bangiales, Rhodophyta)

NASA Astrophysics Data System (ADS)

Liu, Jiao; Li, Xianchao; Tang, Xuexi; Zhou, Bin

2016-03-01

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis ( PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia.

PubMed

Contreras Gutiérrez, María Angélica; Vivero, Rafael J; Vélez, Iván D; Porter, Charles H; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.

DNA Barcoding for the Identification of Sand Fly Species (Diptera, Psychodidae, Phlebotominae) in Colombia

PubMed Central

Contreras Gutiérrez, María Angélica; Vivero, Rafael J.; Vélez, Iván D.; Porter, Charles H.; Uribe, Sandra

2014-01-01

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia. PMID:24454877

DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

PubMed

Mazzanti, Morena; Alessandrini, Federica; Tagliabracci, Adriano; Wells, Jeffrey D; Campobasso, Carlo P

2010-02-25

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments. 2009 Elsevier Ireland Ltd. All rights reserved.

System/observer/controller identification toolbox

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan; Horta, Lucas G.; Phan, Minh

1992-01-01

System Identification is the process of constructing a mathematical model from input and output data for a system under testing, and characterizing the system uncertainties and measurement noises. The mathematical model structure can take various forms depending upon the intended use. The SYSTEM/OBSERVER/CONTROLLER IDENTIFICATION TOOLBOX (SOCIT) is a collection of functions, written in MATLAB language and expressed in M-files, that implements a variety of modern system identification techniques. For an open loop system, the central features of the SOCIT are functions for identification of a system model and its corresponding forward and backward observers directly from input and output data. The system and observers are represented by a discrete model. The identified model and observers may be used for controller design of linear systems as well as identification of modal parameters such as dampings, frequencies, and mode shapes. For a closed-loop system, an observer and its corresponding controller gain directly from input and output data.

Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions

PubMed Central

Zhao, Jun; Kong, Fanrong; Li, Ruoyu; Wang, Xiaohong; Wan, Zhe; Wang, Duanli

2001-01-01

Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens. PMID:11376067

DNA barcoding gap: reliable species identification over morphological and geographical scales.

PubMed

Čandek, Klemen; Kuntner, Matjaž

2015-03-01

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a 'barcoding gap' by comparing intra- and interspecific means, medians and overlap in more than 75,000 computed Kimura 2-parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information. © 2014 John Wiley & Sons Ltd.

Identification of DNA-Binding Proteins Using Structural, Electrostatic and Evolutionary Features

PubMed Central

Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2009-01-01

Summary DNA binding proteins (DBPs) often take part in various crucial processes of the cell's life cycle. Therefore, the identification and characterization of these proteins are of great importance. We present here a random forests classifier for identifying DBPs among proteins with known three-dimensional structures. First, clusters of evolutionarily conserved regions (patches) on the protein's surface are detected using the PatchFinder algorithm; previous studies showed that these regions are typically the proteins' functionally important regions. Next, we train a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein including its dipole moment. Using 10-fold cross validation on a dataset of 138 DNA-binding proteins and 110 proteins which do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of previously published methods. Furthermore, when we tested 5 different methods on 11 new DBPs which did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA. PMID:19233205

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

PubMed Central

Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

2013-01-01

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

PubMed Central

Sonet, Gontran; Jordaens, Kurt; Braet, Yves; Bourguignon, Luc; Dupont, Eréna; Backeljau, Thierry; De Meyer, Marc; Desmyter, Stijn

2013-01-01

Abstract Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes. PMID:24453564

A reliable DNA barcode reference library for the identification of the North European shelf fish fauna.

PubMed

Knebelsberger, Thomas; Landi, Monica; Neumann, Hermann; Kloppmann, Matthias; Sell, Anne F; Campbell, Patrick D; Laakmann, Silke; Raupach, Michael J; Carvalho, Gary R; Costa, Filipe O

2014-09-01

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour-joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within-species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51-fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two-thirds of the typical fish species recorded for the North Sea. © 2014 John Wiley & Sons Ltd.

Identification and reassessment of the specific status of some tropical freshwater midges (Diptera: Chironomidae) using DNA barcode data.

PubMed

Pramual, Pairot; Simwisat, Kusumart; Martin, Jon

2016-01-28

Chironomidae are a highly diverse group of insects. Members of this family are often included in programs monitoring the health of freshwater ecosystems. However, a difficulty in morphological identification, particularly of larval stages is the major obstacle to this application. In this study, we tested the efficiency of mitochondrial cytochrome c oxidase I (COI) sequences as the DNA barcoding region for species identification of Chironomidae in Thailand. The results revealed 14 species with a high success rate (>90%) for the correct species identification, which suggests the potential usefulness of the technique. However, some morphological species possess high (>3%) intraspecific genetic divergence that suggests these species could be species complexes and need further morphological or cytological examination. Sequence-based species delimitation analyses indicated that most specimens identified as Chironomus kiiensis, Tokunaga 1936, in Japan are conspecific with C. striatipennis, Kieffer 1912, although a small number form a separate cluster. A review of the descriptions of Kiefferulus tainanus (Kieffer 1912) and its junior synonym, K. biroi (Kieffer 1918), following our results, suggests that this synonymy is probably not correct and that K. tainanus occurs in Japan, China and Singapore, while K. biroi occurs in India and Thailand. Our results therefore revealed the usefulness of DNA barcoding for correct species identification of Chironomidae, particularly the immature stages. In addition, DNA barcodes could also uncover hidden diversity that can guide further taxonomic study, and offer a more efficient way to identify species than morphological analysis where large numbers of specimens are involved, provided the identifications of DNA barcodes in the databases are correct. Our studies indicate that this is not the case, and we identify cases of misidentifications for C. flaviplumus, Tokunaga 1940 and K. tainanus.

Evaluation of the Biotyper MALDI-TOF MS system for identification of Staphylococcus species.

PubMed

Zhu, Wenming; Sieradzki, Krzysztof; Albrecht, Valerie; McAllister, Sigrid; Lin, Wen; Stuchlik, Olga; Limbago, Brandi; Pohl, Jan; Kamile Rasheed, J

2015-10-01

The Bruker Biotyper MALDI-TOF MS (Biotyper) system, with a modified 30 minute formic acid extraction method, was evaluated by its ability to identify 216 clinical Staphylococcus isolates from the CDC reference collection comprising 23 species previously identified by conventional biochemical tests. 16S rDNA sequence analysis was used to resolve discrepancies. Of these, 209 (96.8%) isolates were correctly identified: 177 (84.7%) isolates had scores ≥2.0, while 32 (15.3%) had scores between 1.70 and 1.99. The Biotyper identification was inconsistent with the biochemical identification for seven (3.2%) isolates, but the Biotyper identifications were confirmed by 16S rDNA analysis. The distribution of low scores was strongly species-dependent, e.g. only 5% of Staphylococcus epidermidis and 4.8% of Staphylococcus aureus isolates scored below 2.0, while 100% of Staphylococcus cohnii, 75% of Staphylococcus sciuri, and 60% of Staphylococcus caprae produced low but accurate Biotyper scores. Our results demonstrate that the Biotyper can reliably identify Staphylococcus species with greater accuracy than conventional biochemicals. Broadening of the reference database by inclusion of additional examples of under-represented species could further optimize Biotyper results. Published by Elsevier B.V.

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

PubMed Central

Kulsantiwong, Jutharat; Prasopdee, Sattrachai; Ruangsittichai, Jiraporn; Ruangjirachuporn, Wipaporn; Boonmars, Thidarut; Viyanant, Vithoon; Pierossi, Paola; Hebert, Paul D. N.; Tesana, Smarn

2013-01-01

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy. PMID:24223896

DNA based identification of medicinal materials in Chinese patent medicines

NASA Astrophysics Data System (ADS)

Chen, Rong; Dong, Juan; Cui, Xin; Wang, Wei; Yasmeen, Afshan; Deng, Yun; Zeng, Xiaomao; Tang, Zhuo

2012-12-01

Chinese patent medicines (CPM) are highly processed and easy to use Traditional Chinese Medicine (TCM). The market for CPM in China alone is tens of billions US dollars annually and some of the CPM are also used as dietary supplements for health augmentation in the western countries. But concerns continue to be raised about the legality, safety and efficacy of many popular CPM. Here we report a pioneer work of applying molecular biotechnology to the identification of CPM, particularly well refined oral liquids and injections. What's more, this PCR based method can also be developed to an easy to use and cost-effective visual chip by taking advantage of G-quadruplex based Hybridization Chain Reaction. This study demonstrates that DNA identification of specific Medicinal materials is an efficient and cost-effective way to audit highly processed CPM and will assist in monitoring their quality and legality.

Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA

PubMed Central

Boyer, Stephane; Brown, Samuel D. J.; Collins, Rupert A.; Cruickshank, Robert H.; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D.

2012-01-01

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation. PMID:22666489

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses.

PubMed

Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

2015-01-01

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

PubMed Central

Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

2015-01-01

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

PubMed Central

Hartvig, Ida; Czako, Mihaly; Kjær, Erik Dahl; Nielsen, Lene Rostgaard; Theilade, Ida

2015-01-01

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and

[Analysis of genetic diversity of Russian regional populations based on common STR markers used in DNA identification].

PubMed

Pesik, V Yu; Fedunin, A A; Agdzhoyan, A T; Utevska, O M; Chukhraeva, M I; Evseeva, I V; Churnosov, M I; Lependina, I N; Bogunov, Yu V; Bogunova, A A; Ignashkin, M A; Yankovsky, N K; Balanovska, E V; Orekhov, V A; Balanovsky, O P

2014-06-01

We conducted the first genetic analysis of a wide a range of rural Russian populations in European Russia with a panel of common DNA markers commonly used in criminalistics genetic identification. We examined a total of 647 samples from indigenous ethnic Russian populations in Arkhangelsk, Belgorod, Voronezh, Kursk, Rostov, Ryazan, and Orel regions. We employed a multiplex genotyping kit, COrDIS Plus, to genotype Short Tandem Repeat (STR) loci, which included the genetic marker panel officially recommended for DNA identification in the Russian Federation, the United States, and the European Union. In the course of our study, we created a database of allelic frequencies, examined the distribution of alleles and genotypes in seven rural Russian populations, and defined the genetic relationships between these populations. We found that, although multidimensional analysis indicated a difference between the Northern gene pool and the rest of the Russian European populations, a pairwise comparison using 19 STR markers among all populations did not reveal significant differences. This is in concordance with previous studies, which examined up to 12 STR markers of urban Russian populations. Therefore, the database of allelic frequencies created in this study can be applied for forensic examinations and DNA identification among the ethnic Russian population over European Russia. We also noted a decrease in the levels of heterozygosity in the northern Russian population compared to ethnic populations in southern and central Russia, which is consistent with trends identified previously using classical gene markers and analysis of mitochondrial DNA.

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

PubMed

Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

2018-04-01

Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

Identification of DNA-binding proteins by combining auto-cross covariance transformation and ensemble learning.

PubMed

Liu, Bin; Wang, Shanyi; Dong, Qiwen; Li, Shumin; Liu, Xuan

2016-04-20

DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. With the rapid development of next generation of sequencing technique, the number of protein sequences is unprecedentedly increasing. Thus it is necessary to develop computational methods to identify the DNA-binding proteins only based on the protein sequence information. In this study, a novel method called iDNA-KACC is presented, which combines the Support Vector Machine (SVM) and the auto-cross covariance transformation. The protein sequences are first converted into profile-based protein representation, and then converted into a series of fixed-length vectors by the auto-cross covariance transformation with Kmer composition. The sequence order effect can be effectively captured by this scheme. These vectors are then fed into Support Vector Machine (SVM) to discriminate the DNA-binding proteins from the non DNA-binding ones. iDNA-KACC achieves an overall accuracy of 75.16% and Matthew correlation coefficient of 0.5 by a rigorous jackknife test. Its performance is further improved by employing an ensemble learning approach, and the improved predictor is called iDNA-KACC-EL. Experimental results on an independent dataset shows that iDNA-KACC-EL outperforms all the other state-of-the-art predictors, indicating that it would be a useful computational tool for DNA binding protein identification. .

Development of biometric DNA ink for authentication security.

PubMed

Hashiyada, Masaki

2004-10-01

Among the various types of biometric personal identification systems, DNA provides the most reliable personal identification. It is intrinsically digital and unchangeable while the person is alive, and even after his/her death. Increasing the number of DNA loci examined can enhance the power of discrimination. This report describes the development of DNA ink, which contains synthetic DNA mixed with printing inks. Single-stranded DNA fragments encoding a personalized set of short tandem repeats (STR) were synthesized. The sequence was defined as follows. First, a decimal DNA personal identification (DNA-ID) was established based on the number of STRs in the locus. Next, this DNA-ID was encrypted using a binary, 160-bit algorithm, using a hashing function to protect privacy. Since this function is irreversible, no one can recover the original information from the encrypted code. Finally, the bit series generated above is transformed into base sequences, and double-stranded DNA fragments are amplified by the polymerase chain reaction (PCR) to protect against physical attacks. Synthesized DNA was detected successfully after samples printed in DNA ink were subjected to several resistance tests used to assess the stability of printing inks. Endurance test results showed that this DNA ink would be suitable for practical use as a printing ink and was resistant to 40 hours of ultraviolet exposure, performance commensurate with that of photogravure ink. Copyright 2004 Tohoku University Medical Press

Identification of DNA-binding proteins using structural, electrostatic and evolutionary features.

PubMed

Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir

2009-04-10

DNA-binding proteins (DBPs) participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance. We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions (patches) on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein, including its dipole moment. Using 10-fold cross-validation on a dataset of 138 DBPs and 110 proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of published methods. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.

28 CFR 28.13 - Analysis and indexing of DNA samples.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau of...

28 CFR 28.13 - Analysis and indexing of DNA samples.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau of...

28 CFR 28.13 - Analysis and indexing of DNA samples.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau of...

28 CFR 28.13 - Analysis and indexing of DNA samples.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau of...

28 CFR 28.13 - Analysis and indexing of DNA samples.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau of...

Map-invariant spectral analysis for the identification of DNA periodicities

PubMed Central

2012-01-01

Many signal processing based methods for finding hidden periodicities in DNA sequences have primarily focused on assigning numerical values to the symbolic DNA sequence and then applying spectral analysis tools such as the short-time discrete Fourier transform (ST-DFT) to locate these repeats. The key results pertaining to this approach are however obtained using a very specific symbolic to numerical map, namely the so-called Voss representation. An important research problem is to therefore quantify the sensitivity of these results to the choice of the symbolic to numerical map. In this article, a novel algebraic approach to the periodicity detection problem is presented and provides a natural framework for studying the role of the symbolic to numerical map in finding these repeats. More specifically, we derive a new matrix-based expression of the DNA spectrum that comprises most of the widely used mappings in the literature as special cases, shows that the DNA spectrum is in fact invariable under all these mappings, and generates a necessary and sufficient condition for the invariance of the DNA spectrum to the symbolic to numerical map. Furthermore, the new algebraic framework decomposes the periodicity detection problem into several fundamental building blocks that are totally independent of each other. Sophisticated digital filters and/or alternate fast data transforms such as the discrete cosine and sine transforms can therefore be always incorporated in the periodicity detection scheme regardless of the choice of the symbolic to numerical map. Although the newly proposed framework is matrix based, identification of these periodicities can be achieved at a low computational cost. PMID:23067324

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

PubMed Central

Langley, Alexander R.; Gräf, Stefan; Smith, James C.; Krude, Torsten

2016-01-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. PMID:27587586

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

PubMed

Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

2016-12-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

PubMed

Furutani, Shunsuke; Hagihara, Yoshihisa; Nagai, Hidenori

2017-09-01

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

PubMed Central

Little, Damon P.

2011-01-01

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types. PMID:21857897

The archaeal Ced system imports DNA

PubMed Central

van Wolferen, Marleen; Wagner, Alexander; van der Does, Chris; Albers, Sonja-Verena

2016-01-01

The intercellular transfer of DNA is a phenomenon that occurs in all domains of life and is a major driving force of evolution. Upon UV-light treatment, cells of the crenarchaeal genus Sulfolobus express Ups pili, which initiate cell aggregate formation. Within these aggregates, chromosomal DNA, which is used for the repair of DNA double-strand breaks, is exchanged. Because so far no clear homologs of bacterial DNA transporters have been identified among the genomes of Archaea, the mechanisms of archaeal DNA transport have remained a puzzling and underinvestigated topic. Here we identify saci_0568 and saci_0748, two genes from Sulfolobus acidocaldarius that are highly induced upon UV treatment, encoding a transmembrane protein and a membrane-bound VirB4/HerA homolog, respectively. DNA transfer assays showed that both proteins are essential for DNA transfer between Sulfolobus cells and act downstream of the Ups pili system. Our results moreover revealed that the system is involved in the import of DNA rather than the export. We therefore propose that both Saci_0568 and Saci_0748 are part of a previously unidentified DNA importer. Given the fact that we found this transporter system to be widely spread among the Crenarchaeota, we propose to name it the Crenarchaeal system for exchange of DNA (Ced). In this study we have for the first time to our knowledge described an archaeal DNA transporter. PMID:26884154

Mitochondrial DNA identification of game and harvested freshwater fish species.

PubMed

Kyle, C J; Wilson, C C

2007-02-14

The use of DNA in forensics has grown rapidly for human applications along with the concomitant development of bioinformatics and demographic databases to help fully realize the potential of this molecular information. Similar techniques are also used routinely in many wildlife cases, such as species identification in food products, poaching and the illegal trade of endangered species. The use of molecular techniques in forensic cases related to wildlife and the development of associated databases has, however, mainly focused on large mammals with the exception of a few high-profile species. There is a need to develop similar databases for aquatic species for fisheries enforcement, given the large number of exploited and endangered fish species, the intensity of exploitation, and challenges in identifying species and their derived products. We sequenced a 500bp fragment of the mitochondrial cytochrome b gene from representative individuals from 26 harvested fish taxa from Ontario, Canada, focusing on species that support major commercial and recreational fisheries. Ontario provides a unique model system for the development of a fish species database, as the province contains an evolutionarily diverse array of freshwater fish families representing more than one third of all freshwater fish in Canada. Inter- and intraspecific sequence comparisons using phylogenetic analysis and a BLAST search algorithm provided rigorous statistical metrics for species identification. This methodology and these data will aid in fisheries enforcement, providing a tool to easily and accurately identify fish species in enforcement investigations that would have otherwise been difficult or impossible to pursue.

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. Methods We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). Results We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). Conclusions This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination

Sorting through the chaff, nDNA gene trees for phylogenetic inference and hybrid identification of annual sunflowers (Helianthus sect. Helianthus).

PubMed

Moody, Michael L; Rieseberg, Loren H

2012-07-01

The annual sunflowers (Helianthus sect. Helianthus) present a formidable challenge for phylogenetic inference because of ancient hybrid speciation, recent introgression, and suspected issues with deep coalescence. Here we analyze sequence data from 11 nuclear DNA (nDNA) genes for multiple genotypes of species within the section to (1) reconstruct the phylogeny of this group, (2) explore the utility of nDNA gene trees for detecting hybrid speciation and introgression; and (3) test an empirical method of hybrid identification based on the phylogenetic congruence of nDNA gene trees from tightly linked genes. We uncovered considerable topological heterogeneity among gene trees with or without three previously identified hybrid species included in the analyses, as well as a general lack of reciprocal monophyly of species. Nonetheless, partitioned Bayesian analyses provided strong support for the reciprocal monophyly of all species except H. annuus (0.89 PP), the most widespread and abundant annual sunflower. Previous hypotheses of relationships among taxa were generally strongly supported (1.0 PP), except among taxa typically associated with H. annuus, apparently due to the paraphyly of the latter in all gene trees. While the individual nDNA gene trees provided a useful means for detecting recent hybridization, identification of ancient hybridization was problematic for all ancient hybrid species, even when linkage was considered. We discuss biological factors that affect the efficacy of phylogenetic methods for hybrid identification.

Detection of Orthopoxvirus DNA by Real-Time PCR and Identification of Variola Virus DNA by Melting Analysis

PubMed Central

Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

2004-01-01

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification. PMID:15004077

Salmon redd identification using environmental DNA (eDNA)

USGS Publications Warehouse

Pilliod, David S.; Laramie, Matthew B.

2016-06-10

IntroductionThe purpose of this project was to develop a technique to use environmental DNA (eDNA) to distinguish between redds made by Chinook salmon (Oncorhynchus tshawytscha) and redds made by Coho salmon (O. kisutch) and to distinguish utilized redds from test/abandoned redds or scours that have the appearance of redds. The project had two phases:Phase 1. Develop, test, and optimize a molecular assay for detecting and identifying Coho salmon DNA and differentiating it from Chinook salmon DNA.Phase 2. Demonstrate the efficacy of the technique.Collect and preserve water samples from the interstitial spaces of 10 known redds (as identified by expert observers) of each species and 10 gravel patches that do not include a redd of either species.Collect control samples from the water column adjacent to each redd to establish background eDNA levels.Analyze the samples using the developed molecular assays for Coho salmon (phase I) and Chinook salmon (Laramie and others, 2015).Evaluate whether samples collected from Chinook and Coho redds have significantly higher levels of eDNA of the respective species than background levels (that is, from gravel, water column).Evaluate whether samples collected from the interstitial spaces of gravel patches that are not redds are similar to background eDNA levels.The Sandy River is a large tributary of the Columbia River. The Sandy River meets the Columbia River approximately 23 km upstream of Portland, Oregon. The Sandy River Basin provides overlapping spawning habitat for both Chinook and Coho salmon.Samples provided by Portland Water Bureau for analysis were collected from the Bull Run River, Sixes Creek, Still Creek, Arrah Wanna Side Channel, and Side Channel 18.

Author Identification Systems

ERIC Educational Resources Information Center

Wagner, A. Ben

2009-01-01

Many efforts are currently underway to disambiguate author names and assign unique identification numbers so that publications by a given scholar can be reliably grouped together. This paper reviews a number of operational and in-development services. Some systems like ResearcherId.Com depend on self-registration and self-identification of a…

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species.

PubMed

Nwani, Christopher D; Becker, Sven; Braid, Heather E; Ude, Emmanuel F; Okogwu, Okechukwu I; Hanner, Robert

2011-10-01

Fishes are the main animal protein source for human beings and play a vital role in aquatic ecosystems and food webs. Fish identification can be challenging, especially in the tropics (due to high diversity), and this is particularly true for larval forms or fragmentary remains. DNA barcoding, which uses the 5' region of the mitochondrial cytochrome c oxidase subunit I (COI) as a target gene, is an efficient method for standardized species-level identification for biodiversity assessment and conservation, pending the establishment of reference sequence libraries. In this study, fishes were collected from three rivers in southeastern Nigeria, identified morphologically, and imaged digitally. DNA was extracted, PCR-amplified, and the standard barcode region was bidirectionally sequenced for 363 individuals belonging to 70 species in 38 genera. All specimen provenance data and associated sequence information were recorded in the barcode of life data systems (BOLD; www.barcodinglife.org ). Analytical tools on BOLD were used to assess the performance of barcoding to identify species. Using neighbor-joining distance comparison, the average genetic distance was 60-fold higher between species than within species, as pairwise genetic distance estimates averaged 10.29% among congeners and only 0.17% among conspecifics. Despite low levels of divergence within species, we observed river system-specific haplotype partitioning within eight species (11.4% of all species). Our preliminary results suggest that DNA barcoding is very effective for species identification of Nigerian freshwater fishes.

Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

PubMed Central

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-01-01

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria. PMID:25884791

Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

PubMed

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-04-15

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

Identification of cephalopod species from the North and Baltic Seas using morphology, COI and 18S rDNA sequences

NASA Astrophysics Data System (ADS)

Gebhardt, Katharina; Knebelsberger, Thomas

2015-09-01

We morphologically analyzed 79 cephalopod specimens from the North and Baltic Seas belonging to 13 separate species. Another 29 specimens showed morphological features of either Alloteuthis mediaor Alloteuthis subulata or were found to be in between. Reliable identification features to distinguish between A. media and A. subulata are currently not available. The analysis of the DNA barcoding region of the COI gene revealed intraspecific distances (uncorrected p) ranging from 0 to 2.13 % (average 0.1 %) and interspecific distances between 3.31 and 22 % (average 15.52 %). All species formed monophyletic clusters in a neighbor-joining analysis and were supported by bootstrap values of ≥99 %. All COI haplotypes belonging to the 29 Alloteuthis specimens were grouped in one cluster. Neither COI nor 18S rDNA sequences helped to distinguish between the different Alloteuthis morphotypes. For species identification purposes, we recommend the use of COI, as it showed higher bootstrap support of species clusters and less amplification and sequencing failure compared to 18S. Our data strongly support the assumption that the genus Alloteuthis is only represented by a single species, at least in the North Sea. It remained unclear whether this species is A. subulata or A. media. All COI sequences including important metadata were uploaded to the Barcode of Life Data Systems and can be used as reference library for the molecular identification of more than 50 % of the cephalopod fauna known from the North and Baltic Seas.

Use of rbcL and trnL-F as a Two-Locus DNA Barcode for Identification of NW-European Ferns: An Ecological Perspective

PubMed Central

de Groot, G. Arjen; During, Heinjo J.; Maas, Jan W.; Schneider, Harald; Vogel, Johannes C.; Erkens, Roy H. J.

2011-01-01

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of

Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

PubMed

Harpke, Doerte; Peterson, Angela

2008-05-01

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae)

PubMed Central

Zou, Shanmei; Fei, Cong; Wang, Chun; Gao, Zhan; Bao, Yachao; He, Meilin; Wang, Changhai

2016-01-01

Microalgae identification is extremely difficult. The efficiency of DNA barcoding in microalgae identification involves ideal gene markers and approaches employed, which however, is still under the way. Although Scenedesmus has obtained much research in producing lipids its identification is difficult. Here we present a comprehensive coalescent, distance and character-based DNA barcoding for 118 Scenedesmus strains based on rbcL, tufA, ITS and 16S. The four genes, and their combined data rbcL + tufA + ITS + 16S, rbcL + tufA and ITS + 16S were analyzed by all of GMYC, P ID, PTP, ABGD, and character-based barcoding respectively. It was apparent that the three combined gene data showed a higher proportion of resolution success than the single gene. In comparison, the GMYC and PTP analysis produced more taxonomic lineages. The ABGD generated various resolution in discrimination among the single and combined data. The character-based barcoding was proved to be the most effective approach for species discrimination in both single and combined data which produced consistent species identification. All the integrated results recovered 11 species, five out of which were revealed as potential cryptic species. We suggest that the character-based DNA barcoding together with other approaches based on multiple genes and their combined data could be more effective in microalgae diversity revelation. PMID:27827440

How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae).

PubMed

Zou, Shanmei; Fei, Cong; Wang, Chun; Gao, Zhan; Bao, Yachao; He, Meilin; Wang, Changhai

2016-11-09

Microalgae identification is extremely difficult. The efficiency of DNA barcoding in microalgae identification involves ideal gene markers and approaches employed, which however, is still under the way. Although Scenedesmus has obtained much research in producing lipids its identification is difficult. Here we present a comprehensive coalescent, distance and character-based DNA barcoding for 118 Scenedesmus strains based on rbcL, tufA, ITS and 16S. The four genes, and their combined data rbcL + tufA + ITS + 16S, rbcL + tufA and ITS + 16S were analyzed by all of GMYC, P ID, PTP, ABGD, and character-based barcoding respectively. It was apparent that the three combined gene data showed a higher proportion of resolution success than the single gene. In comparison, the GMYC and PTP analysis produced more taxonomic lineages. The ABGD generated various resolution in discrimination among the single and combined data. The character-based barcoding was proved to be the most effective approach for species discrimination in both single and combined data which produced consistent species identification. All the integrated results recovered 11 species, five out of which were revealed as potential cryptic species. We suggest that the character-based DNA barcoding together with other approaches based on multiple genes and their combined data could be more effective in microalgae diversity revelation.

Chemocoding as an identification tool where morphological- and DNA-based methods fall short: Inga as a case study.

PubMed

Endara, María-José; Coley, Phyllis D; Wiggins, Natasha L; Forrister, Dale L; Younkin, Gordon C; Nicholls, James A; Pennington, R Toby; Dexter, Kyle G; Kidner, Catherine A; Stone, Graham N; Kursar, Thomas A

2018-04-01

The need for species identification and taxonomic discovery has led to the development of innovative technologies for large-scale plant identification. DNA barcoding has been useful, but fails to distinguish among many species in species-rich plant genera, particularly in tropical regions. Here, we show that chemical fingerprinting, or 'chemocoding', has great potential for plant identification in challenging tropical biomes. Using untargeted metabolomics in combination with multivariate analysis, we constructed species-level fingerprints, which we define as chemocoding. We evaluated the utility of chemocoding with species that were defined morphologically and subject to next-generation DNA sequencing in the diverse and recently radiated neotropical genus Inga (Leguminosae), both at single study sites and across broad geographic scales. Our results show that chemocoding is a robust method for distinguishing morphologically similar species at a single site and for identifying widespread species across continental-scale ranges. Given that species are the fundamental unit of analysis for conservation and biodiversity research, the development of accurate identification methods is essential. We suggest that chemocoding will be a valuable additional source of data for a quick identification of plants, especially for groups where other methods fall short. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Identification of species origin of meat and meat products on the DNA basis: a review.

PubMed

Kumar, Arun; Kumar, Rajiv Ranjan; Sharma, Brahm Deo; Gokulakrishnan, Palanisamy; Mendiratta, Sanjod Kumar; Sharma, Deepak

2015-01-01

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes.

PubMed

Nigro, Lisa M; Angel, Martin V; Blachowiak-Samolyk, Katarzyna; Hopcroft, Russell R; Bucklin, Ann

2016-01-01

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes

PubMed Central

2016-01-01

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes

Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

PubMed Central

Luo, Jun; Cui, Xiuji; Gao, Lu

2017-01-01

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3′ exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3′ end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5′ end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the

Identifications of captive and wild tilapia species existing in Hawaii by mitochondrial DNA control region sequence.

PubMed

Wu, Liang; Yang, Jinzeng

2012-01-01

The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis. In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization. This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species identification. The suspected tilapia hybrids that consist of O. niloticus

Identifications of Captive and Wild Tilapia Species Existing in Hawaii by Mitochondrial DNA Control Region Sequence

PubMed Central

Wu, Liang; Yang, Jinzeng

2012-01-01

Background The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis. Methodology/Principal Findings In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization. Conclusions/Significance This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species

[Identification of Tibetan medicine "Dida" of Gentianaceae using DNA barcoding].

PubMed

Liu, Chuan; Zhang, Yu-Xin; Liu, Yue; Chen, Yi-Long; Fan, Gang; Xiang, Li; Xu, Jiang; Zhang, Yi

2016-02-01

The ITS2 barcode was used toidentify Tibetan medicine "Dida", and tosecure its quality and safety in medication. A total of 13 species, 151 experimental samples for the study from the Tibetan Plateau, including Gentianaceae Swertia, Halenia, Gentianopsis, Comastoma, Lomatogonium ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.7.1. The Kimura 2-Parameter (K2P) distances were calculated using MEGA 6.0. The neighbor-joining (NJ) phylogenetic trees were constructed. There are 31 haplotypes among 231 bp after alignment of all ITS2 sequence haplotypes, and the average G±C content of 61.40%. The NJ tree strongly supported that every species clustered into their own clade and high identification success rate, except that Swertia bifolia and Swertia wolfangiana could not be distinguished from each other based on the sequence divergences. DNA barcoding could be used as a fast and accurate identification method to distinguish Tibetan medicine "Dida" to ensure its safe use. Copyright© by the Chinese Pharmaceutical Association.

Preliminary Identification and Typing of Pathogenic and Toxigenic Fusarium Species Using Restriction Digestion of ITS1-5.8S rDNA-ITS2 Region.

PubMed

Mirhendi, H; Ghiasian, A; Vismer, Hf; Asgary, Mr; Jalalizand, N; Arendrup, Mc; Makimura, K

2010-01-01

Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 and ITS4 universal primers followed by sequencing and restriction enzyme digestion of PCR products. The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species.

Medicinal Plants Recommended by the World Health Organization: DNA Barcode Identification Associated with Chemical Analyses Guarantees Their Quality

PubMed Central

Palhares, Rafael Melo; Gonçalves Drummond, Marcela; dos Santos Alves Figueiredo Brasil, Bruno; Pereira Cosenza, Gustavo; das Graças Lins Brandão, Maria; Oliveira, Guilherme

2015-01-01

Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines. PMID:25978064

33 CFR 401.20 - Automatic Identification System.

Code of Federal Regulations, 2010 CFR

2010-07-01

...' maritime Differential Global Positioning System radiobeacon services; or (7) The use of a temporary unit... Identification System. (a) Each of the following vessels must use an Automatic Identification System (AIS... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Automatic Identification System...

[DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system].

PubMed

Gao, Lin-Lin; Xu, Nian-Lai; Xie, Wei; Ding, Shao-Cheng; Wang, Dong-Jing; Ma, Li-Qin; Li, You-Ying

2013-04-01

To explore a new method in order to extract DNA from bones and teeth automatically. Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system. DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method. AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections.

PubMed

Chambers, E Anne; Hebert, Paul D N

2016-01-01

High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections

PubMed Central

Chambers, E. Anne; Hebert, Paul D. N.

2016-01-01

Background High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. Methodology/Principal Findings This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. Conclusions/Significance This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

NASA Astrophysics Data System (ADS)

Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

2011-06-01

Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

Recovery Based Nanowire Field-Effect Transistor Detection of Pathogenic Avian Influenza DNA

NASA Astrophysics Data System (ADS)

Lin, Chih-Heng; Chu, Chia-Jung; Teng, Kang-Ning; Su, Yi-Jr; Chen, Chii-Dong; Tsai, Li-Chu; Yang, Yuh-Shyong

2012-02-01

Fast and accurate diagnosis is critical in infectious disease surveillance and management. We proposed a DNA recovery system that can easily be adapted to DNA chip or DNA biosensor for fast identification and confirmation of target DNA. This method was based on the re-hybridization of DNA target with a recovery DNA to free the DNA probe. Functionalized silicon nanowire field-effect transistor (SiNW FET) was demonstrated to monitor such specific DNA-DNA interaction using high pathogenic strain virus hemagglutinin 1 (H1) DNA of avian influenza (AI) as target. Specific electric changes were observed in real-time for AI virus DNA sensing and device recovery when nanowire surface of SiNW FET was modified with complementary captured DNA probe. The recovery based SiNW FET biosensor can be further developed for fast identification and further confirmation of a variety of influenza virus strains and other infectious diseases.

PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

PubMed Central

Ristaino, Jean B.; Madritch, Michael; Trout, Carol L.; Parra, Gregory

1998-01-01

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora. PMID:9501434

DNA-based identification of invasive alien species in relation to Canadian federal policy and law, and the basis of rapid-response management.

PubMed

Thomas, Vernon G; Hanner, Robert H; Borisenko, Alex V

2016-11-01

Managing invasive alien species in Canada requires reliable taxonomic identification as the basis of rapid-response management. This can be challenging, especially when organisms are small and lack morphological diagnostic features. DNA-based techniques, such as DNA barcoding, offer a reliable, rapid, and inexpensive toolkit for taxonomic identification of individual or bulk samples, forensic remains, and even environmental DNA. Well suited for this requirement, they could be more broadly deployed and incorporated into the operating policy and practices of Canadian federal departments and should be authorized under these agencies' articles of law. These include Fisheries and Oceans Canada, Canadian Food Inspection Agency, Transport Canada, Environment Canada, Parks Canada, and Health Canada. These efforts should be harmonized with the appropriate provisions of provincial jurisdictions, for example, the Ontario Invasive Species Act. This approach necessitates that a network of accredited, certified laboratories exists, and that updated DNA reference libraries are readily accessible. Harmonizing this approach is vital among Canadian federal agencies, and between the federal and provincial levels of government. Canadian policy and law must also be harmonized with that of the USA when detecting, and responding to, invasive species in contiguous lands and waters. Creating capacity in legislation for use of DNA-based identifications brings the authority to fund, train, deploy, and certify staff, and to refine further developments in this molecular technology.

A molecular identification system for grasses: a novel technology for forensic botany.

PubMed

Ward, J; Peakall, R; Gilmore, S R; Robertson, J

2005-09-10

Our present inability to rapidly, accurately and cost-effectively identify trace botanical evidence remains the major impediment to the routine application of forensic botany. Grasses are amongst the most likely plant species encountered as forensic trace evidence and have the potential to provide links between crime scenes and individuals or other vital crime scene information. We are designing a molecular DNA-based identification system for grasses consisting of several PCR assays that, like a traditional morphological taxonomic key, provide criteria that progressively identify an unknown grass sample to a given taxonomic rank. In a prior study of DNA sequences across 20 phylogenetically representative grass species, we identified a series of potentially informative indels in the grass mitochondrial genome. In this study we designed and tested five PCR assays spanning these indels and assessed the feasibility of these assays to aid identification of unknown grass samples. We confirmed that for our control set of 20 samples, on which the design of the PCR assays was based, the five primer combinations produced the expected results. Using these PCR assays in a 'blind test', we were able to identify 25 unknown grass samples with some restrictions. Species belonging to genera represented in our control set were all correctly identified to genus with one exception. Similarly, genera belonging to tribes in the control set were correctly identified to the tribal level. Finally, for those samples for which neither the tribal or genus specific PCR assays were designed, we could confidently exclude these samples from belonging to certain tribes and genera. The results confirmed the utility of the PCR assays and the feasibility of developing a robust full-scale usable grass identification system for forensic purposes.

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

PubMed Central

2012-01-01

Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. Results This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Conclusions Our results provided fast and cost

Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library.

PubMed

Kuzmina, Maria L; Johnson, Karen L; Barron, Hannah R; Hebert, Paul Dn

2012-11-28

Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Our results provided fast and cost-effective solution to create a

Modal identification of dynamic mechanical systems

NASA Astrophysics Data System (ADS)

Srivastava, R. K.; Kundra, T. K.

1992-07-01

This paper reviews modal identification techniques which are now helping designers all over the world to improve the dynamic behavior of vibrating engineering systems. In this context the need to develop more accurate and faster parameter identification is ever increasing. A new dynamic stiffness matrix based identification method which is highly accurate, fast and system-dynamic-modification compatible is presented. The technique is applicable to all those multidegree-of-freedom systems where full receptance matrix can be experimentally measured.

Genetic identification of missing persons: DNA analysis of human remains and compromised samples.

PubMed

Alvarez-Cubero, M J; Saiz, M; Martinez-Gonzalez, L J; Alvarez, J C; Eisenberg, A J; Budowle, B; Lorente, J A

2012-01-01

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers. Copyright © 2012 S. Karger AG, Basel.

Use of rDNA polymorphism for identification of Heterophyidae infecting freshwater fishes.

PubMed

Dzikowski, R; Levy, M G; Poore, M F; Flowers, J R; Paperna, I

2004-04-21

Infections by trematodes are among the most common fish-borne zoonoses. Metacercariae of the Family Heterophyidae in marine and freshwater fishes are nonfastidious in their choice of definitive hosts, and therefore, cause infections in human and domestic animals. In the present study, species-specific polymerase chain reaction (PCR) assays were developed for identifying and differentiating the various species examined. Sequencing and aligning the 18S (SSU) rDNA revealed interspecific variation for which species-specific DNA oligonucleotides were designed and used for the identification of 6 heterophyid species recovered from piscivorous birds. The oligonucleotides were further used to evaluate the various stages (cercariae recovered from snails, metacercariae recovered from fish and adult trematodes) of the digeneans. By applying this method we elucidated for the first time the life cycle of Pygidiopsis genata. The phylogenetic interrelationship among the newly sequenced species of Heterophyidae is outlined.

DNA Barcoding Reveals Limited Accuracy of Identifications Based on Folk Taxonomy

PubMed Central

Martin, Gary; Abbad, Abdelaziz; Kool, Anneleen

2014-01-01

Background The trade of plant roots as traditional medicine is an important source of income for many people around the world. Destructive harvesting practices threaten the existence of some plant species. Harvesters of medicinal roots identify the collected species according to their own folk taxonomies, but once the dried or powdered roots enter the chain of commercialization, accurate identification becomes more challenging. Methodology A survey of morphological diversity among four root products traded in the medina of Marrakech was conducted. Fifty-one root samples were selected for molecular identification using DNA barcoding using three markers, trnH-psbA, rpoC1, and ITS. Sequences were searched using BLAST against a tailored reference database of Moroccan medicinal plants and their closest relatives submitted to NCBI GenBank. Principal Findings Combining psbA-trnH, rpoC1, and ITS allowed the majority of the market samples to be identified to species level. Few of the species level barcoding identifications matched the scientific names given in the literature, including the most authoritative and widely cited pharmacopeia. Conclusions/Significance The four root complexes selected from the medicinal plant products traded in Marrakech all comprise more than one species, but not those previously asserted. The findings have major implications for the monitoring of trade in endangered plant species as morphology-based species identifications alone may not be accurate. As a result, trade in certain species may be overestimated, whereas the commercialization of other species may not be recorded at all. PMID:24416210

Parameter identification for nonlinear aerodynamic systems

NASA Technical Reports Server (NTRS)

Pearson, Allan E.

1991-01-01

Work continues on frequency analysis for transfer function identification, both with respect to the continued development of the underlying algorithms and in the identification study of two physical systems. Some new results of a theoretical nature were recently obtained that lend further insight into the frequency domain interpretation of the research. Progress in each of those areas is summarized. Although not related to the system identification problem, some new results were obtained on the feedback stabilization of linear time lag systems.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine.

PubMed

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li; Li, Haifeng

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine.

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine

PubMed Central

Yang, Fan; Ding, Fei; Chen, Hong; He, Mingqi; Zhu, Shixin; Ma, Xin; Jiang, Li

2018-01-01

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine. PMID:29849709

Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs.

PubMed

Campbell, A J; Gasser, R B; Chilton, N B

1995-03-01

In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.

Evaluation of Automated Yeast Identification System

NASA Technical Reports Server (NTRS)

McGinnis, M. R.

1996-01-01

One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

Gender-Associated Mitochondrial DNA Heteroplasmy in Somatic Tissues of the Endangered Freshwater Mussel Unio crassus (Bivalvia: Unionidae): Implications for Sex Identification and Phylogeographical Studies.

PubMed

Mioduchowska, Monika; Kaczmarczyk, Agnieszka; Zając, Katarzyna; Zając, Tadeusz; Sell, Jerzy

2016-11-01

Some bivalve species possess two independent mitochondrial DNA lineages: maternally (F-type) and paternally (M-type) inherited. This phenomenon is called doubly uniparental inheritance. It is generally agreed that F-type mtDNA is typically present in female somatic and gonadal tissues as well as in male somatic tissues, whereas the M-type mtDNA occurs only in male germ line and gonadal tissue. In the present study, the mtDNA heteroplasmy (for both F and M genomes) in male somatic tissues of Unio crassus (Philipsson, 1788), species threatened with extinction, has been confirmed. Taking advantage from the presence of Mcox1 marker only in male somatic tissues, we developed a new method of sex identification in this endangered species, using nondestructive tissue sampling. Probability of correct sex identification was estimated at 97.5%. The present study is the first report on gender-associated mitochondrial DNA heteroplasmy in male somatic tissues of thick-shelled river mussel and first approach to U. crassus sex identification at molecular level. Our study also confirmed the utility of paternally inherited Mcox1 gene fragment as a complementary molecular tool for resolving phylogeographical relationships among populations of thick-shelled river mussel. © 2017 Wiley Periodicals, Inc.

A universal DNA-based protein detection system.

PubMed

Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

2013-09-25

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

A Universal DNA-Based Protein Detection System

PubMed Central

Tran, Thua N. N.; Cui, Jinhui; Hartman, Mark R.; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C.; Lis, John T.; Cui, Haixin; Luo, Dan

2014-01-01

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability. PMID:23978265

Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects.

PubMed

Bannwarth, Sylvie; Procaccio, Vincent; Paquis-Flucklinger, Veronique

2005-06-01

Molecular analysis of mitochondrial DNA (mtDNA) is a critical step in diagnosis and genetic counseling of respiratory chain defects. No fast method is currently available for the identification of unknown mtDNA point mutations. We have developed a new strategy based on complete mtDNA PCR amplification followed by digestion with a mismatch-specific DNA endonuclease, Surveyor Nuclease. This enzyme, a member of the CEL nuclease family of plant DNA endonucleases, cleaves double-strand DNA at any mismatch site including base substitutions and small insertions/deletions. After digestion, cleavage products are separated and analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. Although this method allows the analysis of 2 kb mtDNA amplicons and the detection of multiple mutations within the same fragment, it does not lead to the identification of homoplasmic base substitutions. Homoplasmic pathogenic mutations have been described. Nevertheless, most homoplasmic base substitutions are neutral polymorphisms while deleterious mutations are typically heteroplasmic. Here, we report that this method can be used to detect mtDNA mutations such as m.3243A>G tRNA(Leu) and m.14709T>C tRNA(Glu) even when they are present at levels as low as 3% in DNA samples derived from patients with respiratory chain defects. Then, we tested five patients suffering from a mitochondrial respiratory chain defect and we identified a variant (m.16189T>C) in two of them, which was previously associated with susceptibility to diabetes and cardiomyopathy. In conclusion, this method can be effectively used to rapidly and completely screen the entire human mitochondrial genome for heteroplasmic mutations and in this context represents an important advance for the diagnosis of mitochondrial diseases.

Decentralized System Identification Using Stochastic Subspace Identification for Wireless Sensor Networks

PubMed Central

Cho, Soojin; Park, Jong-Woong; Sim, Sung-Han

2015-01-01

Wireless sensor networks (WSNs) facilitate a new paradigm to structural identification and monitoring for civil infrastructure. Conventional structural monitoring systems based on wired sensors and centralized data acquisition systems are costly for installation as well as maintenance. WSNs have emerged as a technology that can overcome such difficulties, making deployment of a dense array of sensors on large civil structures both feasible and economical. However, as opposed to wired sensor networks in which centralized data acquisition and processing is common practice, WSNs require decentralized computing algorithms to reduce data transmission due to the limitation associated with wireless communication. In this paper, the stochastic subspace identification (SSI) technique is selected for system identification, and SSI-based decentralized system identification (SDSI) is proposed to be implemented in a WSN composed of Imote2 wireless sensors that measure acceleration. The SDSI is tightly scheduled in the hierarchical WSN, and its performance is experimentally verified in a laboratory test using a 5-story shear building model. PMID:25856325

Identification of DNA Methyltransferase Genes in Human Pathogenic Bacteria by Comparative Genomics.

PubMed

Brambila-Tapia, Aniel Jessica Leticia; Poot-Hernández, Augusto Cesar; Perez-Rueda, Ernesto; Rodríguez-Vázquez, Katya

2016-06-01

DNA methylation plays an important role in gene expression and virulence in some pathogenic bacteria. In this report, we describe DNA methyltransferases (MTases) present in human pathogenic bacteria and compared them with related species, which are not pathogenic or less pathogenic, based in comparative genomics. We performed a search in the KEGG database of the KEGG database orthology groups associated with adenine and cytosine DNA MTase activities (EC: 2.1.1.37, EC: 2.1.1.113 and EC: 2.1.1.72) in 37 human pathogenic species and 18 non/less pathogenic relatives and performed comparisons of the number of these MTases sequences according to their genome size, the DNA MTase type and with their non-less pathogenic relatives. We observed that Helicobacter pylori and Neisseria spp. presented the highest number of MTases while ten different species did not present a predicted DNA MTase. We also detected a significant increase of adenine MTases over cytosine MTases (2.19 vs. 1.06, respectively, p < 0.001). Adenine MTases were the only MTases associated with restriction modification systems and DNA MTases associated with type I restriction modification systems were more numerous than those associated with type III restriction modification systems (0.84 vs. 0.17, p < 0.001); additionally, there was no correlation with the genome size and the total number of DNA MTases, indicating that the number of DNA MTases is related to the particular evolution and lifestyle of specific species, regulating the expression of virulence genes in some pathogenic bacteria.

Automation and validation of DNA-banking systems.

PubMed

Thornton, Melissa; Gladwin, Amanda; Payne, Robin; Moore, Rachael; Cresswell, Carl; McKechnie, Douglas; Kelly, Steve; March, Ruth

2005-10-15

DNA banking is one of the central capabilities on which modern genetic research rests. The DNA-banking system plays an essential role in the flow of genetic data from patients and genetics researchers to the application of genetic research in the clinic. Until relatively recently, large collections of DNA samples were not common in human genetics. Now, collections of hundreds of thousands of samples are common in academic institutions and private companies. Automation of DNA banking can dramatically increase throughput, eliminate manual errors and improve the productivity of genetics research. An increased emphasis on pharmacogenetics and personalized medicine has highlighted the need for genetics laboratories to operate within the principles of a recognized quality system such as good laboratory practice (GLP). Automated systems are suitable for such laboratories but require a level of validation that might be unfamiliar to many genetics researchers. In this article, we use the AstraZeneca automated DNA archive and reformatting system (DART) as a case study of how such a system can be successfully developed and validated within the principles of GLP.

Calibrating snakehead diversity with DNA barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species.

PubMed

Serrao, Natasha R; Steinke, Dirk; Hanner, Robert H

2014-01-01

Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.

Identification of DNA-binding proteins using multi-features fusion and binary firefly optimization algorithm.

PubMed

Zhang, Jian; Gao, Bo; Chai, Haiting; Ma, Zhiqiang; Yang, Guifu

2016-08-26

DNA-binding proteins (DBPs) play fundamental roles in many biological processes. Therefore, the developing of effective computational tools for identifying DBPs is becoming highly desirable. In this study, we proposed an accurate method for the prediction of DBPs. Firstly, we focused on the challenge of improving DBP prediction accuracy with information solely from the sequence. Secondly, we used multiple informative features to encode the protein. These features included evolutionary conservation profile, secondary structure motifs, and physicochemical properties. Thirdly, we introduced a novel improved Binary Firefly Algorithm (BFA) to remove redundant or noisy features as well as select optimal parameters for the classifier. The experimental results of our predictor on two benchmark datasets outperformed many state-of-the-art predictors, which revealed the effectiveness of our method. The promising prediction performance on a new-compiled independent testing dataset from PDB and a large-scale dataset from UniProt proved the good generalization ability of our method. In addition, the BFA forged in this research would be of great potential in practical applications in optimization fields, especially in feature selection problems. A highly accurate method was proposed for the identification of DBPs. A user-friendly web-server named iDbP (identification of DNA-binding Proteins) was constructed and provided for academic use.

Application of DNA-based methods in forensic entomology.

PubMed

Wells, Jeffrey D; Stevens, Jamie R

2008-01-01

A forensic entomological investigation can benefit from a variety of widely practiced molecular genotyping methods. The most commonly used is DNA-based specimen identification. Other applications include the identification of insect gut contents and the characterization of the population genetic structure of a forensically important insect species. The proper application of these procedures demands that the analyst be technically expert. However, one must also be aware of the extensive list of standards and expectations that many legal systems have developed for forensic DNA analysis. We summarize the DNA techniques that are currently used in, or have been proposed for, forensic entomology and review established genetic analyses from other scientific fields that address questions similar to those in forensic entomology. We describe how accepted standards for forensic DNA practice and method validation are likely to apply to insect evidence used in a death or other forensic entomological investigation.

[Molecular identification and detection of moon jellyfish (Aurelia sp.) based on partial sequencing of mitochondrial 16S rDNA and COI].

PubMed

Wang, Jian-Yan; Zhen, Yu; Wang, Guo-shan; Mi, Tie-Zhu; Yu, Zhi-gang

2013-03-01

Taking the moon jellyfish Aurelia sp. commonly found in our coastal sea areas as test object, its genome DNA was extracted, the partial sequences of mt-16S rDNA (650 bp) and mt-COI (709 bp) were PCR-amplified, and, after purification, cloning, and sequencing, the sequences obtained were BLASTn-analyzed. The sequences of greater difference with those of the other jellyfish were chosen, and eight specific primers for the mt-16S rDNA and mt-COI of Aurelia sp. were designed, respectively. The specificity test indicated that the primer AS3 for the mt-16S rDNA and the primer AC3 for the mt-COI were excellent in rapidly detecting the target jellyfish from Rhopilema esculentum, Nemopilema nomurai, Cyanea nozakii, Acromitus sp., and Aurelia sp., and thus, the techniques for the molecular identification and detection of moon jellyfish were preliminarily established, which could get rid of the limitations in classical morphological identification of Aurelia sp. , being able to find the Aurelia sp. in the samples more quickly and accurately.

Validation of DNA-based identification software by computation of pedigree likelihood ratios.

PubMed

Slooten, K

2011-08-01

Disaster victim identification (DVI) can be aided by DNA-evidence, by comparing the DNA-profiles of unidentified individuals with those of surviving relatives. The DNA-evidence is used optimally when such a comparison is done by calculating the appropriate likelihood ratios. Though conceptually simple, the calculations can be quite involved, especially with large pedigrees, precise mutation models etc. In this article we describe a series of test cases designed to check if software designed to calculate such likelihood ratios computes them correctly. The cases include both simple and more complicated pedigrees, among which inbred ones. We show how to calculate the likelihood ratio numerically and algebraically, including a general mutation model and possibility of allelic dropout. In Appendix A we show how to derive such algebraic expressions mathematically. We have set up these cases to validate new software, called Bonaparte, which performs pedigree likelihood ratio calculations in a DVI context. Bonaparte has been developed by SNN Nijmegen (The Netherlands) for the Netherlands Forensic Institute (NFI). It is available free of charge for non-commercial purposes (see www.dnadvi.nl for details). Commercial licenses can also be obtained. The software uses Bayesian networks and the junction tree algorithm to perform its calculations. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao).

PubMed

Tian, Enwei; Liu, Qianqian; Ye, Haoting; Li, Fang; Chao, Zhi

2017-12-18

Background: Wuzhimaotao (the dry root of Ficus hirta ) is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans , which contains gelsemine that is extremely neurotoxic) and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA) with species-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min) of the ITS region under a constant and mild temperature range of 37-42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fields, C.A.

1996-06-01

The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progressmore » report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.« less

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

PubMed

Pryce, Todd M; Palladino, Silvano; Price, Diane M; Gardam, Dianne J; Campbell, Peter B; Christiansen, Keryn J; Murray, Ronan J

2006-04-01

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

Developing an Apicomplexan DNA Barcoding System to Detect Blood Parasites of Small Coral Reef Fishes.

PubMed

Renoux, Lance P; Dolan, Maureen C; Cook, Courtney A; Smit, Nico J; Sikkel, Paul C

2017-08-01

Apicomplexan parasites are obligate parasites of many species of vertebrates. To date, there is very limited understanding of these parasites in the most-diverse group of vertebrates, actinopterygian fishes. While DNA barcoding targeting the eukaryotic 18S small subunit rRNA gene sequence has been useful in identifying apicomplexans in tetrapods, identification of apicomplexans infecting fishes has relied solely on morphological identification by microscopy. In this study, a DNA barcoding method was developed that targets the 18S rRNA gene primers for identifying apicomplexans parasitizing certain actinopterygian fishes. A lead primer set was selected showing no cross-reactivity to the overwhelming abundant host DNA and successfully confirmed 37 of the 41 (90.2%) microscopically verified parasitized fish blood samples analyzed in this study. Furthermore, this DNA barcoding method identified 4 additional samples that screened negative for parasitemia, suggesting this molecular method may provide improved sensitivity over morphological characterization by microscopy. In addition, this PCR screening method for fish apicomplexans, using Whatman FTA preserved DNA, was tested in efforts leading to a more simplified field collection, transport, and sample storage method as well as a streamlining sample processing important for DNA barcoding of large sample sets.

gene GIS: Computational Tools for Spatial Analyses of DNA Profiles with Associated Photo-Identification and Telemetry Records of Marine Mammals

DTIC Science & Technology

2011-09-30

DNA profiles. Referred to as geneGIS, the program will provide the ability to display, browse, select, filter and summarize spatial or temporal...of the SPLASH photo-identification records and available DNA profiles is underway through integration and crosschecking by Cascadia and MMI . An...Darwin Core standards where possible and can accommodate the current databases developed for telemetry data at MMI and SPLASH collection records at

A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

PubMed Central

2010-01-01

Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification. PMID:21062433

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae).

PubMed

Syromyatnikov, Mikhail Y; Golub, Victor B; Kokina, Anastasia V; Victoria A Soboleva; Popov, Vasily N

2017-01-01

The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps . Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps , E. maura , and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura , E. testudinarius , E. dilaticollis , could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps , the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps .

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae)

PubMed Central

Syromyatnikov, Mikhail Y.; Golub, Victor B.; Kokina, Anastasia V.; Victoria A. Soboleva; Popov, Vasily N.

2017-01-01

Abstract The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps. Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps, E. maura, and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura, E. testudinarius, E. dilaticollis, could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps, the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps. PMID:29118620

DNA Barcodes for Species Identification in the Hyperdiverse Ant Genus Pheidole (Formicidae: Myrmicinae)

PubMed Central

Ng'endo, R.N.; Osiemo, Z.B.; Brandl, R.

2013-01-01

DNA sequencing is increasingly being used to assist in species identification in order to overcome taxonomic impediment. However, few studies attempt to compare the results of these molecular studies with a more traditional species delineation approach based on morphological characters. Mitochondrial DNA Cytochrome oxidase subunit 1 (CO1) gene was sequenced, measuring 636 base pairs, from 47 ants of the genus Pheidole (Formicidae: Myrmicinae) collected in the Brazilian Atlantic Forest to test whether the morphology-based assignment of individuals into species is supported by DNA-based species delimitation. Twenty morphospecies were identified, whereas the barcoding analysis identified 19 Molecular Operational Taxonomic Units (MOTUs). Fifteen out of the 19 DNA-based clusters allocated, using sequence divergence thresholds of 2% and 3%, matched with morphospecies. Both thresholds yielded the same number of MOTUs. Only one MOTU was successfully identified to species level using the CO1 sequences of Pheidole species already in the Genbank. The average pairwise sequence divergence for all 47 sequences was 19%, ranging between 0–25%. In some cases, however, morphology and molecular based methods differed in their assignment of individuals to morphospecies or MOTUs. The occurrence of distinct mitochondrial lineages within morphological species highlights groups for further detailed genetic and morphological studies, and therefore a pluralistic approach using several methods to understand the taxonomy of difficult lineages is advocated. PMID:23902257

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

PubMed

Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

2001-03-01

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

Stochastic system identification in structural dynamics

USGS Publications Warehouse

Safak, Erdal

1988-01-01

Recently, new identification methods have been developed by using the concept of optimal-recursive filtering and stochastic approximation. These methods, known as stochastic identification, are based on the statistical properties of the signal and noise, and do not require the assumptions of current methods. The criterion for stochastic system identification is that the difference between the recorded output and the output from the identified system (i.e., the residual of the identification) should be equal to white noise. In this paper, first a brief review of the theory is given. Then, an application of the method is presented by using ambient vibration data from a nine-story building.

Performance of two MALDI-TOF MS systems for the identification of yeasts isolated from bloodstream infections and cerebrospinal fluids using a time-saving direct transfer protocol.

PubMed

Hamprecht, Axel; Christ, Sara; Oestreicher, Tanja; Plum, Georg; Kempf, Volkhard A J; Göttig, Stephan

2014-04-01

The rapid and correct identification of pathogens is of paramount importance for the treatment of patients with invasive infections. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can speed up the identification of bacteria and fungi and has quickly been embraced by medical microbiology laboratories worldwide. Different MALDI-TOF systems have been compared in studies focussing on identification rates of different pathogens. Another aspect that has not been systematically assessed is the performance in daily routine and handling, which is important especially for microbiology routine laboratories. We compared two widespread commercial systems, Microflex LT Biotyper (Bruker) and VitekMS (bioMérieux), for the identification of 210 relevant clinical yeasts under routine conditions, using a time-saving direct transfer protocol. We assessed the need for an additional extraction step, the threshold for species identification and the duration of measurements with the two systems. The tested yeasts included 34 Candida albicans isolates, 144 non-albicans Candida spp. and 32 yeasts of different genera. The results of the two MS systems were compared with that of biochemical identification and, in case of discrepancies, DNA sequencing of the internal transcribed spacer or the large subunit of ribosomal DNA. Both systems correctly identified 96.2 % of isolates [202/210, non-significant (n.s.)]. Misidentifications were observed for VitekMS only (n = 5, no major errors, n.s.). VitekMS was the slower system (19.8 vs. 8.0 min for 10 samples, p = 0.002) but had the advantage of a more effective direct transfer protocol with less need for an additional extraction step.

Performance characterization of material identification systems

NASA Astrophysics Data System (ADS)

Brown, Christopher D.; Green, Robert L.

2006-10-01

In recent years a number of analytical devices have been proposed and marketed specifically to enable field-based material identification. Technologies reliant on mass, near- and mid-infrared, and Raman spectroscopies are available today, and other platforms are imminent. These systems tend to perform material recognition based on an on-board library of material signatures. While figures of merit for traditional quantitative analytical sensors are broadly established (e.g., SNR, selectivity, sensitivity, limit of detection/decision), measures of performance for material identification systems have not been systematically discussed. In this paper we present an approach to performance characterization similar in spirit to ROC curves, but including elements of precision-recall curves and specialized for the intended-use of material identification systems. Important experimental considerations are discussed, including study design, sources of bias, uncertainty estimation, and cross-validation and the approach as a whole is illustrated using a commercially available handheld Raman material identification system.

DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market.

PubMed

Mishra, Priyanka; Kumar, Amit; Nagireddy, Akshitha; Mani, Daya N; Shukla, Ashutosh K; Tiwari, Rakesh; Sundaresan, Velusamy

2016-01-01

The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

[Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding].

PubMed

Wang, Xi; Hou, Fei-xia; Wang, Yi-xuan; Wang, Yu-xian; Li, Jun-de; Yuan, Yuan; Peng, Cheng; Guo, Jin-lin

2015-10-01

Both market research and literature reports both found that the ootheca of mantodea was all used as medicine. However, Chinese Pharmacopoeia only records the ootheca of three mantis species. The clinical use of ootheca unrecorded in Chinese Pharmacopoeia, will pose potential risks to drug safety. It's urgent to identify the origin of Mantidis Oötheca. The current researches about original animal in Mantidis Oötheca are based on morphology and unanimous. DNA barcoding fill gaps of the traditional morphological identification, which is widely used in animal classification studies. This study first use DNA barcoding to analyze genetic distance among different Mantidis Oötheca types, align COI sequences between mantis and Mantidis Oötheca and construct the phylogeny tree. The result confirmed that Tenodera sinensis and Hierodula patellifera were the origin insects of Tuanpiaoxiao and Heipiaoxiao, respectively, and Statilia maculate and Mantis religiosa were the origin insects of Changpiaoxiao.

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

PubMed Central

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

NASA Astrophysics Data System (ADS)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta'Ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-07-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.

Nuclear DNA markers for identification of Beluga and Sterlet sturgeons and their interspecific Bester hybrid.

PubMed

Havelka, Miloš; Fujimoto, Takafumi; Hagihara, Seishi; Adachi, Shinji; Arai, Katsutoshi

2017-05-10

Sturgeons (Acipenseriformes) are among the most endangered species in the world due to fragmentation and destruction of their natural habitats and to overexploitation, mainly for highly priced caviar. This has led to the development of sturgeon culture, originally for reintroduction, but more recently for caviar production. In both cases, accurate species identification is essential. We report a new tool for accurate identification of Huso huso and Acipenser ruthenus based on nuclear DNA markers. We employed ddRAD sequencing to identify species-specific nucleotide variants, which served as specific binding sites for diagnostic primers. The primers allowed identification of Huso huso and Acipenser ruthenus as well as their discrimination from A. baerii, A. schrenckii, A. gueldenstaedtii, A. stellatus, A. persicus, A. mikadoi, A. transmontanus, and H. dauricus and identification of A. ruthenus and H. huso hybrids with these species, except hybrid between A. ruthenus and A. stellatus. The species-specific primers also allowed identification of bester (H. huso × A. ruthenus), the most commercially exploited sturgeon hybrid. The tool, based on simple PCR and gel electrophoresis, is rapid, inexpensive, and reproducible. It will contribute to conservation of remaining wild populations of A. ruthenus and H. huso, as well as to traceability of their products.

DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

ERIC Educational Resources Information Center

Millard, Julie T

2011-01-01

The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

DNA Barcoding for Efficient Species- and Pathovar-Level Identification of the Quarantine Plant Pathogen Xanthomonas

PubMed Central

Tian, Qian; Zhao, Wenjun; Lu, Songyu; Zhu, Shuifang; Li, Shidong

2016-01-01

Genus Xanthomonas comprises many economically important plant pathogens that affect a wide range of hosts. Indeed, fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria for imports in China. To date, however, a rapid and accurate method capable of identifying all of the quarantine species/pathovars has yet to be developed. In this study, we therefore evaluated the capacity of DNA barcoding as a digital identification method for discriminating quarantine species/pathovars of Xanthomonas. For these analyses, 327 isolates, representing 45 Xanthomonas species/pathovars, as well as five additional species/pathovars from GenBank (50 species/pathovars total), were utilized to test the efficacy of four DNA barcode candidate genes (16S rRNA gene, cpn60, gyrB, and avrBs2). Of these candidate genes, cpn60 displayed the highest rate of PCR amplification and sequencing success. The tree-building (Neighbor-joining), ‘best close match’, and barcode gap methods were subsequently employed to assess the species- and pathovar-level resolution of each gene. Notably, all isolates of each quarantine species/pathovars formed a monophyletic group in the neighbor-joining tree constructed using the cpn60 sequences. Moreover, cpn60 also demonstrated the most satisfactory results in both barcoding gap analysis and the ‘best close match’ test. Thus, compared with the other markers tested, cpn60 proved to be a powerful DNA barcode, providing a reliable and effective means for the species- and pathovar-level identification of the quarantine plant pathogen Xanthomonas. PMID:27861494

Assessment of Multi Fragment Melting Analysis System (MFMAS) for the Identification of Food-Borne Yeasts.

PubMed

Kesmen, Zülal; Büyükkiraz, Mine E; Özbekar, Esra; Çelik, Mete; Özkök, F Özge; Kılıç, Özge; Çetin, Bülent; Yetim, Hasan

2018-06-01

Multi Fragment Melting Analysis System (MFMAS) is a novel approach that was developed for the species-level identification of microorganisms. It is a software-assisted system that performs concurrent melting analysis of 8 different DNA fragments to obtain a fingerprint of each strain analyzed. The identification is performed according to the comparison of these fingerprints with the fingerprints of known yeast species recorded in a database to obtain the best possible match. In this study, applicability of the yeast version of the MFMAS (MFMAS-yeast) was evaluated for the identification of food-associated yeast species. For this purpose, in this study, a total of 145 yeast strains originated from foods and beverages and 19 standard yeast strains were tested. The DNAs isolated from these yeast strains were analyzed by the MFMAS, and their species were successfully identified with a similarity rate of 95% or higher. It was shown that the strains belonged to 43 different yeast species that are widely found in the foods. A clear discrimination was also observed in the phylogenetically related species. In conclusion, it might be suggested that the MFMAS-yeast seems to be a highly promising approach for a rapid, accurate, and one-step identification of the yeasts isolated from food products and/or their processing environments.

Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

PubMed

Branda, John A; Rychert, Jenna; Burnham, Carey-Ann D; Bythrow, Maureen; Garner, Omai B; Ginocchio, Christine C; Jennemann, Rebecca; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Sercia, Linda F; Westblade, Lars F; Ferraro, Mary Jane

2014-02-01

The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level. © 2013.

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling.

PubMed

Calacal, Gayvelline C; Delfin, Frederick C; Tan, Michelle Music M; Roewer, Lutz; Magtanong, Danilo L; Lara, Myra C; Fortun, Raquel dR; De Ungria, Maria Corazon A

2005-09-01

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.

DNA-based identification of Calendula officinalis (Asteraceae)1

PubMed Central

Schmiderer, Corinna; Lukas, Brigitte; Ruzicka, Joana; Novak, Johannes

2015-01-01

Premise of the study: For the economically important species Calendula officinalis, a fast identification assay based on high-resolution melting curve analysis was designed. This assay was developed to distinguish C. officinalis from other species of the genus and other Asteraceae genera, and to detect C. officinalis as an adulterant of saffron samples. Methods and Results: For this study, five markers (ITS, rbcL, 5′ trnK-matK, psbA-trnH, trnL-trnF) of 10 Calendula species were sequenced and analyzed for species-specific mutations. With the application of two developed primer pairs located in the trnK 5′ intron and trnL-trnF, C. officinalis could be distinguished from other species of the genus and all outgroup samples tested. Adulterations of Calendula DNA in saffron could be detected down to 0.01%. Conclusions: With the developed assay, C. officinalis can be reliably identified and admixtures of this species as adulterant of saffron can be revealed at low levels. PMID:26649268

Identification of the major tamoxifen-DNA adducts in rat liver by mass spectroscopy.

PubMed

Rajaniemi, H; Rasanen, I; Koivisto, P; Peltonen, K; Hemminki, K

1999-02-01

We present here the first mass spectroscopic (MS) identification of the main tamoxifen-induced DNA adducts in rat liver. The two main adducts were isolated by high-performance liquid chromatography (HPLC) and identified by MS, MS-MS and ultraviolet spectroscopy. Adduct 1 was the N-desmethyltamoxifen-deoxyguanosine adduct in which the alpha-position of the metabolite N-desmethyltamoxifen is linked covalently to the amino group of deoxyguanosine. Adduct 2 was confirmed to be the trans isomer of alpha-(N2-deoxyguanosinyl)tamoxifen, as previously suggested by co-chromatography.

Decentralized system identification using stochastic subspace identification on wireless smart sensor networks

NASA Astrophysics Data System (ADS)

Sim, Sung-Han; Spencer, Billie F., Jr.; Park, Jongwoong; Jung, Hyungjo

2012-04-01

Wireless Smart Sensor Networks (WSSNs) facilitates a new paradigm to structural identification and monitoring for civil infrastructure. Conventional monitoring systems based on wired sensors and centralized data acquisition and processing have been considered to be challenging and costly due to cabling and expensive equipment and maintenance costs. WSSNs have emerged as a technology that can overcome such difficulties, making deployment of a dense array of sensors on large civil structures both feasible and economical. However, as opposed to wired sensor networks in which centralized data acquisition and processing is common practice, WSSNs require decentralized computing algorithms to reduce data transmission due to the limitation associated with wireless communication. Thus, several system identification methods have been implemented to process sensor data and extract essential information, including Natural Excitation Technique with Eigensystem Realization Algorithm, Frequency Domain Decomposition (FDD), and Random Decrement Technique (RDT); however, Stochastic Subspace Identification (SSI) has not been fully utilized in WSSNs, while SSI has the strong potential to enhance the system identification. This study presents a decentralized system identification using SSI in WSSNs. The approach is implemented on MEMSIC's Imote2 sensor platform and experimentally verified using a 5-story shear building model.

Choosing and Using a Plant DNA Barcode

PubMed Central

Hollingsworth, Peter M.; Graham, Sean W.; Little, Damon P.

2011-01-01

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance. PMID:21637336

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

PubMed

Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

2008-10-01

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

Species-specific identification from incomplete sampling: applying DNA barcodes to monitoring invasive solanum plants.

PubMed

Zhang, Wei; Fan, Xiaohong; Zhu, Shuifang; Zhao, Hong; Fu, Lianzhong

2013-01-01

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling-through this, DNA barcoding will greatly benefit the current fields of its application.

DNA barcoding and development of species-specific markers for the identification of tea mosquito bugs (Miridae: Heteroptera) in India.

PubMed

Rebijith, K B; Asokan, R; Kumar, N K Krishna; Srikumar, K K; Ramamurthy, V V; Bhat, P Shivarama

2012-10-01

Rapid, accurate, and timely identification of insects as a group is important and challenging worldwide, as they outnumber all other animals in number and diversity. DNA barcoding is a method for the identification of species in a wide range of animal taxa, which uses the 5' region of the mitochondrial cytochrome c oxidase-I (CO-I). Yet another easy, accurate, and economical method of species discrimination is by developing species-specific markers, which produce specific amplicon for the species in question. The method is handy because it is not limited by life stages, sex, polymorphism, and other factors. Herein, we measured the usefulness of CO-I for the species discrimination of mirids in India viz. Helopeltis antonii Signoret, H. thievora Waterhouse, H. bradyi Waterhouse, and Pachypeltis maesarum Kirkaldy in their various life stages. Furthermore, our study showed the utility of species-specific markers in differentiating H. antonii (295) and H. bradyi (514) regardless of their life stages. Analysis of CO-I gene revealed <1% intraspecific divergence for all four species examined, whereas the interspecific distances ranged from 7 to 13%. This study showed that the DNA barcode and species-specific markers will aid the identification of mirids in India and will stand as a decisive tool in formulating integrated pest management (IPM) strategy, quick identification of invasive and cryptic species, haplotypes, biotypes, and other factors, if any.

Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

PubMed

Poovitha, Sundar; Stalin, Nithaniyal; Balaji, Raju; Parani, Madasamy

2016-12-01

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes rbcLa and matK together with the suggested additional regions trnH-psbA and ITS2. No intraspecies divergence was observed among the accessions studied. Interspecies divergence was 0%-9.6% with individual markers, which increased to 0%-12.5% and 0.8%-20.3% when using two- and three-marker combinations, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when the matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.

Effectiveness of ITS and sub-regions as DNA barcode markers for the identification of Basidiomycota (Fungi).

PubMed

Badotti, Fernanda; de Oliveira, Francislon Silva; Garcia, Cleverson Fernando; Vaz, Aline Bruna Martins; Fonseca, Paula Luize Camargos; Nahum, Laila Alves; Oliveira, Guilherme; Góes-Neto, Aristóteles

2017-02-23

Fungi are among the most abundant and diverse organisms on Earth. However, a substantial amount of the species diversity, relationships, habitats, and life strategies of these microorganisms remain to be discovered and characterized. One important factor hindering progress is the difficulty in correctly identifying fungi. Morphological and molecular characteristics have been applied in such tasks. Later, DNA barcoding has emerged as a new method for the rapid and reliable identification of species. The nrITS region is considered the universal barcode of Fungi, and the ITS1 and ITS2 sub-regions have been applied as metabarcoding markers. In this study, we performed a large-scale analysis of all the available Basidiomycota sequences from GenBank. We carried out a rigorous trimming of the initial dataset based in methodological principals of DNA Barcoding. Two different approaches (PCI and barcode gap) were used to determine the performance of the complete ITS region and sub-regions. For most of the Basidiomycota genera, the three genomic markers performed similarly, i.e., when one was considered a good marker for the identification of a genus, the others were also; the same results were observed when the performance was insufficient. However, based on barcode gap analyses, we identified genomic markers that had a superior identification performance than the others and genomic markers that were not indicated for the identification of some genera. Notably, neither the complete ITS nor the sub-regions were useful in identifying 11 of the 113 Basidiomycota genera. The complex phylogenetic relationships and the presence of cryptic species in some genera are possible explanations of this limitation and are discussed. Knowledge regarding the efficiency and limitations of the barcode markers that are currently used for the identification of organisms is crucial because it benefits research in many areas. Our study provides information that may guide researchers in choosing

An overview of recent advances in system identification

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan

1994-01-01

This paper presents an overview of the recent advances in system identification for modal testing and control of large flexible structures. Several techniques are discussed including the Observer/Kalman Filter Identification, the Observer/Controller Identification, and the State-Space System Identification in the Frequency Domain. The System/Observer/Controller Toolbox developed at NASA Langley Research Center is used to show the applications of these techniques to real aerospace structures such as the Hubble spacecraft telescope and the active flexible aircraft wing.

A brief review of machine vision in the context of automated wood identification systems

Treesearch

John C. Hermanson; Alex C. Wiedenhoeft

2011-01-01

The need for accurate and rapid field identification of wood to combat illegal logging around the world is outpacing the ability to train personnel to perform this task. Despite increased interest in non-anatomical (DNA, spectroscopic, chemical) methods for wood identification, anatomical characteristics are the least labile data that can be extracted from solid wood...

DNA barcoding for identification of sand fly species (Diptera: Psychodidae) from leishmaniasis-endemic areas of Peru.

PubMed

Nzelu, Chukwunonso O; Cáceres, Abraham G; Arrunátegui-Jiménez, Martín J; Lañas-Rosas, Máximo F; Yañez-Trujillano, Henrry H; Luna-Caipo, Deysi V; Holguín-Mauricci, Carlos E; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

2015-05-01

Phlebotomine sand flies are the only proven vectors of leishmaniases, a group of human and animal diseases. Accurate knowledge of sand fly species identification is essential in understanding the epidemiology of leishmaniasis and vector control in endemic areas. Classical identification of sand fly species based on morphological characteristics often remains difficult and requires taxonomic expertise. Here, we generated DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene using 159 adult specimens morphologically identified to be 19 species of sand flies, belonging to 6 subgenera/species groups circulating in Peru, including the vector species. Neighbor-joining (NJ) analysis based on Kimura 2-Parameter genetic distances formed non-overlapping clusters for all species. The levels of intraspecific genetic divergence ranged from 0 to 5.96%, whereas interspecific genetic divergence among different species ranged from 8.39 to 19.08%. The generated COI barcodes could discriminate between all the sand fly taxa. Besides its success in separating known species, we found that DNA barcoding is useful in revealing population differentiation and cryptic diversity, and thus promises to be a valuable tool for epidemiological studies of leishmaniasis. Copyright © 2015 Elsevier B.V. All rights reserved.

30 CFR 75.1715 - Identification check system.

Code of Federal Regulations, 2011 CFR

2011-07-01

... system. [Statutory Provisions] Each operator of a coal mine shall establish a check-in and check-out system which will provide positive identification of every person underground, and will provide an... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Identification check system. 75.1715 Section 75...

30 CFR 75.1715 - Identification check system.

Code of Federal Regulations, 2012 CFR

2012-07-01

... system. [Statutory Provisions] Each operator of a coal mine shall establish a check-in and check-out system which will provide positive identification of every person underground, and will provide an... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Identification check system. 75.1715 Section 75...

30 CFR 75.1715 - Identification check system.

Code of Federal Regulations, 2010 CFR

2010-07-01

... system. [Statutory Provisions] Each operator of a coal mine shall establish a check-in and check-out system which will provide positive identification of every person underground, and will provide an... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Identification check system. 75.1715 Section 75...

30 CFR 75.1715 - Identification check system.

Code of Federal Regulations, 2013 CFR

2013-07-01

... system. [Statutory Provisions] Each operator of a coal mine shall establish a check-in and check-out system which will provide positive identification of every person underground, and will provide an... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Identification check system. 75.1715 Section 75...

30 CFR 75.1715 - Identification check system.

Code of Federal Regulations, 2014 CFR

2014-07-01

... system. [Statutory Provisions] Each operator of a coal mine shall establish a check-in and check-out system which will provide positive identification of every person underground, and will provide an... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Identification check system. 75.1715 Section 75...

Comparison of methods for the identification of microorganisms isolated from blood cultures.

PubMed

Monteiro, Aydir Cecília Marinho; Fortaleza, Carlos Magno Castelo Branco; Ferreira, Adriano Martison; Cavalcante, Ricardo de Souza; Mondelli, Alessandro Lia; Bagagli, Eduardo; da Cunha, Maria de Lourdes Ribeiro de Souza

2016-08-05

Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.

DNA barcoding using skin exuviates can improve identification and biodiversity studies of snakes.

PubMed

Khedkar, Trupti; Sharma, Rashmi; Tiknaik, Anita; Khedkar, Gulab; Naikwade, Bhagwat S; Ron, Tetsuzan Benny; Haymer, David

2016-01-01

Snakes represent a taxonomically underdeveloped group of animals in India with a lack of experts and incomplete taxonomic descriptions being the main deterrents to advances in this area. Molecular taxonomic approaches using DNA barcoding could aid in snake identification as well as studies of biodiversity. Here a non-invasive sampling method using DNA barcoding is tested using skin exuviates. Taxonomically authenticated samples were collected and tested for validation and comparisons to unknown snake exuviate samples. This approach was also used to construct the first comprehensive study targeting the snake species from Maharashtra state in India. A total of 92 skin exuviate samples were collected and tested for this study. Of these, 81 samples were successfully DNA barcoded and compared with unknown samples for assignment of taxonomic identity. Good quality DNA was obtained irrespective of age and quality of the exuviate material, and all unknown samples were successfully identified. A total of 23 species of snakes were identified, six of which were in the list of Endangered species (Red Data Book). Intra- and inter-specific distance values were also calculated, and these were sufficient to allow discrimination among species and between species without ambiguity in most cases. Two samples were suspected to represent cryptic species based on deep K2P divergence values (>3%), and one sample could be identified to the genus level only. Eleven samples failed to amplify COI sequences, suggesting the need for alternative PCR primer pairs. This study clearly documents how snake skin exuviates can be used for DNA barcoding, estimates of diversity and population genetic structuring in a noninvasive manner.

DNA-based identification of forensically important species of Sarcophagidae (Insecta: Diptera) from Rio de Janeiro, Brazil.

PubMed

Napoleão, K S; Mello-Patiu, C A; Oliveira-Costa, J; Takiya, D M; Silva, R; Moura-Neto, R S

2016-05-06

Sarcophagidae, or flesh flies, are of great importance in forensic entomology, but their effective application requires precise taxonomic identification, which relies almost exclusively on characteristics of the male genitalia. Given that female flies and larvae are most abundant in animal carcasses or on corpses, precise morphological identification can be difficult; therefore, DNA sequencing can be an additional tool for use in taxonomic identification. This paper analyzes part of the mitochondrial cytochrome c oxidase subunit I (COI) gene from three Sarcophagidae species of forensic importance in the City of Rio de Janeiro: Oxysarcodexia fluminensis, Peckia chrysostoma, and Peckia intermutans. COI fragments of 400 bp from 36 specimens of these three species were sequenced. No intraspecific differences were found among specimens of O. fluminensis, but P. chrysostoma and P. intermutans each had two haplotypes, ranging from 0 to 0.7%. The interspecific divergence was 8.5-11.6%, corroborating previously reported findings.

Analysis of DNA from post-blast pipe bomb fragments for identification and determination of ancestry.

PubMed

Tasker, Esiri; LaRue, Bobby; Beherec, Charity; Gangitano, David; Hughes-Stamm, Sheree

2017-05-01

Improvised explosive devices (IEDs) such as pipe bombs are weapons used to detrimentally affect people and communities. A readily accessible brand of exploding targets called Tannerite® has been identified as a potential material for abuse as an explosive in pipe bombs. The ability to recover and genotype DNA from such weapons may be vital in the effort to identify suspects associated with these devices. While it is possible to recover DNA from post-blast fragments using short tandem repeat markers (STRs), genotyping success can be negatively affected by low quantities of DNA, degradation, and/or PCR inhibitors. Alternative markers such as insertion/null (INNULs) and single nucleotide polymorphisms (SNPs) are bi-allelic genetic markers that are shorter genomic targets than STRs for amplification, which are more likely to resist degradation. In this study, we constructed pipe bombs that were spiked with known amounts of biological material to: 1) recover "touch" DNA from the surface of the device, and 2) recover traces of blood from the ends of wires (simulated finger prick). The bombs were detonated with the binary explosive Tannerite® using double-base smokeless powder to initiate the reaction. DNA extracted from the post-blast fragments was quantified with the Quantifiler® Trio DNA Quantification Kit. STR analysis was conducted using the GlobalFiler® Amplification Kit, INNULs were amplified using an early-access version of the InnoTyper™ 21 Kit, and SNP analysis via massively parallel sequencing (MPS) was performed using the HID-Ion Ampliseq™ Identity and Ancestry panels using the Ion Chef and Ion PGM sequencing system. The results of this study showed that INNUL markers resulted in the most complete genetic profiles when compared to STR and SNP profiles. The random match probabilities calculated for samples using INNULs were lower than with STRs when less than 14 STR alleles were reported. These results suggest that INNUL analysis may be well suited for

Disaster victim identification of military aircrew, 1945-2002.

PubMed

Smith, Adrian

2003-11-01

Aviation accident fatalities are characterized by substantial tissue disruption and fragmentation, limiting the usefulness of traditional identification methods. This study examines the success of disaster victim identification (DVI) in military aviation accident fatalities in the Australian Defense Force (ADF). Accident reports and autopsy records of aircrew fatalities during the period 1945-2002 were examined to identify difficulties experienced during the DVI process or injuries that would prevent identification of remains using non-DNA methods. The ADF had 301 aircraft fatalities sustained in 144 accidents during the period 1945-2002. The autopsy reports for 117 fatalities were reviewed (covering 73.7% of aircrew fatalities from 1960-2002). Of the 117 victims, 38 (32.4%) sustained injuries which were severe enough to prevent identification by traditional (non-DNA) comparative scientific DVI techniques of fingerprint and dental analysis. Many of the ADF fatalities who could not be positively identified in the past could be identified today through the use of DNA techniques. Successful DNA identification, however, depends on having a reference DNA profile. This paper recommends the establishment of a DNA repository to store reference blood samples to facilitate the identification of ADF aircrew remains without causing additional distress to family members.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

Integrating early detection with DNA barcoding: species identification of a non-native monitor lizard (Squamata: Varanidae) carcass in Mississippi, U.S.A.

USGS Publications Warehouse

Reed, Robert N.; Hopken, Matthew W.; Steen, David A.; Falk, Bryan G.; Piaggio, Antoinette J.

2016-01-01

Early detection of invasive species is critical to increasing the probability of successful management. At the primary stage of an invasion, invasive species are easier to control as the population is likely represented by just a few individuals. Detection of these first few individuals can be challenging, particularly if they are cryptic or otherwise characterized by low detectability. The engagement of members of the public may be critical to early detection as there are far more citizen s on the landscape than trained biologists. However, it can be difficult to assess the credibility of public reporting, especially when a diagnostic digital image or a physical specimen in good condition are lacking. DNA barcoding can be used for verification when morphological identification of a specimen is not possible or uncertain (i.e., degraded or partial specimen). DNA barcoding relies on obtaining a DNA sequence from a relatively small fragment of mitochondrial DNA and comparing it to a database of sequences containing a variety of expertly identified species. He rein we report the successful identification of a degraded specimen of a non-native, potentially invasive reptile species (Varanus niloticus) via DNA barcoding, after discovery and reporting by a member of the public.

Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding.

PubMed

Nithaniyal, Stalin; Vassou, Sophie Lorraine; Poovitha, Sundar; Raju, Balaji; Parani, Madasamy

2017-02-01

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

FISH-BOL and seafood identification: geographically dispersed case studies reveal systemic market substitution across Canada.

PubMed

Hanner, Robert; Becker, Sven; Ivanova, Natalia V; Steinke, Dirk

2011-10-01

The Fish Barcode of Life campaign involves a broad international collaboration among scientists working to advance the identification of fishes using DNA barcodes. With over 25% of the world's known ichthyofauna currently profiled, forensic identification of seafood products is now feasible and is becoming routine. Driven by growing consumer interest in the food supply, investigative reporters from five different media establishments procured seafood samples (n = 254) from numerous retail establishments located among five Canadian metropolitan areas between 2008 and 2010. The specimens were sent to the Canadian Centre for DNA Barcoding for analysis. By integrating the results from these individual case studies in a summary analysis, we provide a broad perspective on seafood substitution across Canada. Barcodes were recovered from 93% of the samples (n = 236), and identified using the Barcode of Life Data Systems "species identification" engine ( www.barcodinglife.org ). A 99% sequence similarity threshold was employed as a conservative matching criterion for specimen identification to the species level. Comparing these results against the Canadian Food Inspection Agency's "Fish List" a guideline to interpreting "false, misleading or deceptive" names (as per s 27 of the Fish Inspection regulations) demonstrated that 41% of the samples were mislabeled. Most samples were readily identified; however, this was not true in all cases because some samples had no close match. Others were ambiguous due to limited barcode resolution (or imperfect taxonomy) observed within a few closely related species complexes. The latter cases did not significantly impact the results because even the partial resolution achieved was sufficient to demonstrate mislabeling. This work highlights the functional utility of barcoding for the identification of diverse market samples. It also demonstrates how barcoding serves as a bridge linking scientific nomenclature with approved market names

QPSO-Based Adaptive DNA Computing Algorithm

PubMed Central

Karakose, Mehmet; Cigdem, Ugur

2013-01-01

DNA (deoxyribonucleic acid) computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO). Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1) parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2) adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3) numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm. PMID:23935409

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes

PubMed Central

YUAN, QING-JUN; ZHANG, BIN; JIANG, DAN; ZHANG, WEN-JING; LIN, TSAI-YUN; WANG, NIAN-HE; CHIOU, SHU-JIAU; HUANG, LU-QI

2015-01-01

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes. PMID:24961287

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes.

PubMed

Yuan, Qing-Jun; Zhang, Bin; Jiang, Dan; Zhang, Wen-Jing; Lin, Tsai-Yun; Wang, Nian-He; Chiou, Shu-Jiau; Huang, Lu-Qi

2015-03-01

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes. © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

[Integrated DNA barcoding database for identifying Chinese animal medicine].

PubMed

Shi, Lin-Chun; Yao, Hui; Xie, Li-Fang; Zhu, Ying-Jie; Song, Jing-Yuan; Zhang, Hui; Chen, Shi-Lin

2014-06-01

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India.

PubMed

Dhar, Bishal; Ghosh, Sankar Kumar

2017-09-05

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application. Copyright © 2017 Elsevier B.V. All rights reserved.

Laser mass spectrometry for DNA fingerprinting for forensic applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, C.H.; Tang, K.; Taranenko, N.I.

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals.more » DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.« less

Multicenter Evaluation of the Vitek MS v3.0 System for the Identification of Filamentous Fungi.

PubMed

Rychert, Jenna; Slechta, E Sue; Barker, Adam P; Miranda, Edwin; Babady, N Esther; Tang, Yi-Wei; Gibas, Connie; Wiederhold, Nathan; Sutton, DeAnna; Hanson, Kimberly E

2018-02-01

Invasive fungal infections are an important cause of morbidity and mortality affecting primarily immunocompromised patients. While fungal identification to the species level is critical to providing appropriate therapy, it can be slow and laborious and often relies on subjective morphological criteria. The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has the potential to speed up and improve the accuracy of identification. In this multicenter study, we evaluated the accuracy of the Vitek MS v3.0 system in identifying 1,601 clinical mold isolates compared to identification by DNA sequence analysis and supported by morphological and phenotypic testing. Among the 1,519 isolates representing organisms in the v3.0 database, 91% ( n = 1,387) were correctly identified to the species level. An additional 27 isolates (2%) were correctly identified to the genus level. Fifteen isolates were incorrectly identified, due to either a single incorrect identification ( n = 13) or multiple identifications from different genera ( n = 2). In those cases, when a single identification was provided that was not correct, the misidentification was within the same genus. The Vitek MS v3.0 was unable to identify 91 (6%) isolates, despite repeat testing. These isolates were distributed among all the genera. When considering all isolates tested, even those that were not represented in the database, the Vitek MS v3.0 provided a single correct identification 98% of the time. These findings demonstrate that the Vitek MS v3.0 system is highly accurate for the identification of common molds encountered in the clinical mycology laboratory. Copyright © 2018 American Society for Microbiology.

Identification and DNA annotation of a plasmid isolated from Chromobacterium violaceum.

PubMed

Lima, Daniel C; Nyberg, Lena K; Westerlund, Fredrik; Batistuzzo de Medeiros, Silvia R

2018-03-28

Chromobacterium violaceum is a ß-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium.

Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

NASA Astrophysics Data System (ADS)

Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

DNA Microarray for Rapid Detection and Identification of Food and Water Borne Bacteria: From Dry to Wet Lab.

PubMed

Ranjbar, Reza; Behzadi, Payam; Najafi, Ali; Roudi, Raheleh

2017-01-01

A rapid, accurate, flexible and reliable diagnostic method may significantly decrease the costs of diagnosis and treatment. Designing an appropriate microarray chip reduces noises and probable biases in the final result. The aim of this study was to design and construct a DNA Microarray Chip for a rapid detection and identification of 10 important bacterial agents. In the present survey, 10 unique genomic regions relating to 10 pathogenic bacterial agents including Escherichia coli (E.coli), Shigella boydii, Sh.dysenteriae, Sh.flexneri, Sh.sonnei, Salmonella typhi, S.typhimurium, Brucella sp., Legionella pneumophila, and Vibrio cholera were selected for designing specific long oligo microarray probes. For this reason, the in-silico operations including utilization of the NCBI RefSeq database, Servers of PanSeq and Gview, AlleleID 7.7 and Oligo Analyzer 3.1 was done. On the other hand, the in-vitro part of the study comprised stages of robotic microarray chip probe spotting, bacterial DNAs extraction and DNA labeling, hybridization and microarray chip scanning. In wet lab section, different tools and apparatus such as Nexterion® Slide E, Qarray mini spotter, NimbleGen kit, TrayMix TM S4, and Innoscan 710 were used. A DNA microarray chip including 10 long oligo microarray probes was designed and constructed for detection and identification of 10 pathogenic bacteria. The DNA microarray chip was capable to identify all 10 bacterial agents tested simultaneously. The presence of a professional bioinformatician as a probe designer is needed to design appropriate multifunctional microarray probes to increase the accuracy of the outcomes.

World War One Italian and Austrian soldier identification project: DNA results of the first case.

PubMed

Piccinini, Andrea; Coco, Stefania; Parson, Walther; Cattaneo, Cristina; Gaudio, Daniel; Barbazza, Renzo; Galassi, Andrea

2010-10-01

We report the results of an attempt to identify the supposed remains of a famous World War I (WWI) Italian soldier who was killed in battle along the Italian front in 1915. Thanks to the availability of offspring from both paternal and maternal lineage Y-STRs and mtDNA were analysed and both showed a clear exclusion scenario: the remains did not belong to the supposed war hero. This is the first effort of identification of the remains of soldiers who perished during World War I within a multidisciplinary project aimed at the retrieval of historical and cultural aspects linked to WWI, and the systematic study of the remains of soldiers and ultimately their identification. This last step involves both Italian and Austrian laboratories. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

System for tamper identification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bobbitt, III, John Thomas; Weeks, George E.

2017-09-05

A system for tamper identification. A fastener has a tamper identification surface with a unique grain structure that is altered if the fastener is removed or otherwise exposed to sufficient torque. After a period of time such as e.g., shipment and/or storage of the sealed container, a determination of whether tampering has occurred can be undertaken by examining the grain structure to determine if it has changed since the fastener was used to seal the container or secure the device.

Screening for Protein-DNA Interactions by Automatable DNA-Protein Interaction ELISA

PubMed Central

Schüssler, Axel; Kolukisaoglu, H. Üner; Koch, Grit; Wallmeroth, Niklas; Hecker, Andreas; Thurow, Kerstin; Zell, Andreas; Harter, Klaus; Wanke, Dierk

2013-01-01

DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding

Evaluating the statistical power of DNA-based identification, exemplified by 'The missing grandchildren of Argentina'.

PubMed

Kling, Daniel; Egeland, Thore; Piñero, Mariana Herrera; Vigeland, Magnus Dehli

2017-11-01

Methods and implementations of DNA-based identification are well established in several forensic contexts. However, assessing the statistical power of these methods has been largely overlooked, except in the simplest cases. In this paper we outline general methods for such power evaluation, and apply them to a large set of family reunification cases, where the objective is to decide whether a person of interest (POI) is identical to the missing person (MP) in a family, based on the DNA profile of the POI and available family members. As such, this application closely resembles database searching and disaster victim identification (DVI). If parents or children of the MP are available, they will typically provide sufficient statistical evidence to settle the case. However, if one must resort to more distant relatives, it is not a priori obvious that a reliable conclusion is likely to be reached. In these cases power evaluation can be highly valuable, for instance in the recruitment of additional family members. To assess the power in an identification case, we advocate the combined use of two statistics: the Probability of Exclusion, and the Probability of Exceedance. The former is the probability that the genotypes of a random, unrelated person are incompatible with the available family data. If this is close to 1, it is likely that a conclusion will be achieved regarding general relatedness, but not necessarily the specific relationship. To evaluate the ability to recognize a true match, we use simulations to estimate exceedance probabilities, i.e. the probability that the likelihood ratio will exceed a given threshold, assuming that the POI is indeed the MP. All simulations are done conditionally on available family data. Such conditional simulations have a long history in medical linkage analysis, but to our knowledge this is the first systematic forensic genetics application. Also, for forensic markers mutations cannot be ignored and therefore current models and

Continuous-Time Bilinear System Identification

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan

2003-01-01

The objective of this paper is to describe a new method for identification of a continuous-time multi-input and multi-output bilinear system. The approach is to make judicious use of the linear-model properties of the bilinear system when subjected to a constant input. Two steps are required in the identification process. The first step is to use a set of pulse responses resulting from a constant input of one sample period to identify the state matrix, the output matrix, and the direct transmission matrix. The second step is to use another set of pulse responses with the same constant input over multiple sample periods to identify the input matrix and the coefficient matrices associated with the coupling terms between the state and the inputs. Numerical examples are given to illustrate the concept and the computational algorithm for the identification method.

IDENTIFICATION OF STEROCHEMICAL CONFIGERATION OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8

EPA Science Inventory

The definitive identification of stereochemical configurations of DNA adducts detected by 32P-postlabeling requires co-chromatography of adducts with synthetic chromatographic standards. Four major and several minor DNA adducts are formed by cyclopenta[cd]pyrene (CPP) in strain A...

DNA barcode goes two-dimensions: DNA QR code web server.

PubMed

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

DNA Barcode Goes Two-Dimensions: DNA QR Code Web Server

PubMed Central

Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, “DNA barcode” actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications. PMID:22574113

Specific PCR Identification between Peucedanum praeruptorum and Angelica decursiva and Identification between Them and Adulterant Using DNA Barcode.

PubMed

Han, Bang-Xing; Yuan, Yuan; Huang, Lu-Qi; Zhao, Qun; Tan, Ling-Ling; Song, Xiang-Wen; He, Xiao-Mei; Xu, Tao; Liu, Feng; Wang, Jian

2017-01-01

The traditional Chinese medicine (TCM) Qianhu and Zihuaqianhu are the dried roots of Peucedanum praeruptorum and Angelica decursiva , respectively. Since the plant sources of Qianhu and Zihuaqianhu are more complex, the chemical compositions of P. praeruptorum and A. decursiva are significantly different, and many adulterants exist because of the differences in traditional understanding and medication habits. Therefore, the rapid and accurate identification methods are required. The aim was to study the feasibility of using DNA barcoding to distinguish between Traditional Chinese medicine Qianhu ( Peucedanum praeruptorum ), Zihuaqianhu ( Angelica decursiva ), and common adulterants, based on internal transcribed spacer (ITS) sequences, as well as specific PCR identification between P. praeruptorum and A. decursiva . The ITS sequences of P. praeruptorum , A. decursiva , and adulterant were studied, and a phylogenetic tree was constructed. Based on the ITS barcode, the specific PCR primer pairs QH-CP19s/QH-CP19a and ZHQH-CP3s/ZHQH-CP3a were designed for P. praeruptorum and A. decursiva , respectively. The amplification conditions were optimized, and specific PCR products were obtained. The results showed that the phylogenetic trees constructed using the BI and MP methods were consistent, and P. praeruptorum and A. decursiva sequence haplotypes formed their own monophyly. The experimental results showed that in PCR products, the target bands appeared in the genuine drug and not in the adulterant, which suggests the high specificity of the two primer pairs. The ITS sequence was ideal DNA barcode to identify P. praeruptorum , A. decursiva , and adulterant. The specific PCR is a quick and effective method to distinguish between P. praeruptorum and A. decursiva . Peucedanum praeruptorum and Angelica decursiva sequence haplotypes formed their own monophyly.The ITS sequence was ideal DNA barcode to identify P. praeruptorum , A. decursiva , and adulterant.Specific PCR is a

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources.

PubMed

Lim, Jeongheui; Kim, Sang-Yoon; Kim, Sungmin; Eo, Hae-Seok; Kim, Chang-Bae; Paek, Woon Kee; Kim, Won; Bhak, Jong

2009-12-03

DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org.

Fungal pathogen (mis-) identifications: a case study with DNA barcodes on Melampsora rusts of aspen and white poplar.

PubMed

Feau, Nicolas; Vialle, Agathe; Allaire, Mathieu; Tanguay, Philippe; Joly, David L; Frey, Pascal; Callan, Brenda E; Hamelin, Richard C

2009-01-01

Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial-telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.

Evolution and perspectives of cultivar identification and traceability from tree to oil and table olives by means of DNA markers.

PubMed

Pasqualone, Antonella; Montemurro, Cinzia; di Rienzo, Valentina; Summo, Carmine; Paradiso, Vito Michele; Caponio, Francesco

2016-08-01

In recent years, an increasing number of typicality marks has been awarded to high-quality olive oils produced from local cultivars. In this case, quality control requires effective varietal checks of the starting materials. Moreover, accurate cultivar identification is essential in vegetative-propagated plants distributed by nurseries and is a pre-requisite to register new cultivars. Food genomics provides many tools for cultivar identification and traceability from tree to oil and table olives. The results of the application of different classes of DNA markers to olive with the purpose of checking cultivar identity and variability of plant material are extensively discussed in this review, with special regard to repeatability issues and polymorphism degree. The characterization of olive germplasm from all countries of the Mediterranean basin and from less studied geographical areas is described and innovative high-throughput molecular tools to manage reference collections are reviewed. Then the transferability of DNA markers to processed products - virgin olive oils and table olives - is overviewed to point out strengths and weaknesses, with special regard to (i) the influence of processing steps and storage time on the quantity and quality of residual DNA, (ii) recent advances to overcome the bottleneck of DNA extraction from processed products, (iii) factors affecting whole comparability of DNA profiles between fresh plant materials and end-products, (iv) drawbacks in the analysis of multi-cultivar versus single-cultivar end-products and (v) the potential of quantitative polymerase chain reaction (PCR)-based techniques. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

PubMed

Handy, Sara M; Deeds, Jonathan R; Ivanova, Natalia V; Hebert, Paul D N; Hanner, Robert H; Ormos, Andrea; Weigt, Lee A; Moore, Michelle M; Yancy, Haile F

2011-01-01

The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use.

On Markov parameters in system identification

NASA Technical Reports Server (NTRS)

Phan, Minh; Juang, Jer-Nan; Longman, Richard W.

1991-01-01

A detailed discussion of Markov parameters in system identification is given. Different forms of input-output representation of linear discrete-time systems are reviewed and discussed. Interpretation of sampled response data as Markov parameters is presented. Relations between the state-space model and particular linear difference models via the Markov parameters are formulated. A generalization of Markov parameters to observer and Kalman filter Markov parameters for system identification is explained. These extended Markov parameters play an important role in providing not only a state-space realization, but also an observer/Kalman filter for the system of interest.

DNA typing for personal identification of urine after long-term preservation for testing in doping control.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Ueki, Makoto

2017-08-01

When the tampering of a urine sample is suspected in doping control, personal identification of the sample needs to be determined by short tandem repeat (STR) analysis using DNA. We established a method for extracting DNA from urine samples stored at -20 °C without using any additives or procedures, which is consistent with how samples are required to be managed for doping control. The method, using the Puregene® Blood Core kit followed by NucleoSpin® gDNA Clean-up or NucleoSpin® gDNA Clean-up XS kit, does not need any special instrument and can provide a purified extract with high-quality DNA from up to 40 mL of urine suitable for STR analysis using an AmpFlSTR® Identifiler® PCR amplification kit. Storing urine at -20 °C is detrimental to the stability of DNA. The DNA concentration of preserved urine could not be predicted by specific gravity or creatinine level at the time of urine collection. The DNA concentration of a purified extract (10 μL) was required to be >0.06 ng/μL to ensure a successful STR analysis. Thus, the required extraction volumes of urine preserved for 3-7 years at -20 °C were estimated to be 30 mL and 20 mL to succeed in at least 86% of men and 91% of women, respectively. Considering the long half-life of DNA during long-term preservation, our extraction method is applicable to urine samples stored even for 10 years, which is currently the storage duration allowed (increased from 8 years) before re-examination in doping control. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

PubMed

Hargreaves, P; Rahman, S; Guthrie, P; Taanman, J W; Leonard, J V; Land, J M; Heales, S J R

2002-02-01

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

Identification and molecular epidemiology of dermatophyte isolates by repetitive-sequence-PCR-based DNA fingerprinting using the DiversiLab system in Turkey.

PubMed

Koc, A Nedret; Atalay, Mustafa A; Inci, Melek; Sariguzel, Fatma M; Sav, Hafize

2017-05-01

Dermatophyte species, isolation and identification in clinical samples are still difficult and take a long time. The identification and molecular epidemiology of dermatophytes commonly isolated in a clinical laboratory in Turkey by repetitive sequence-based PCR (rep-PCR) were assessed by comparing the results with those of reference identification. A total of 44 dermatophytes isolated from various clinical specimens of 20 patients with superficial mycoses in Kayseri and 24 patients in Hatay were studied. The identification of dermatophyte isolates was based on the reference identification and rep-PCR using the DiversiLab System (BioMerieux). The genotyping of dermatophyte isolates from different patients was determined by rep-PCR. In the identification of dermatophyte isolates, agreement between rep-PCR and conventional methods was 87.8 % ( 36 of 41). The dermatophyte strains belonged to four clones (A -D) which were determined by the use of rep-PCR. The dermatophyte strains in Clone B, D showed identical patterns with respect to the region. In conclusion, rep-PCR appears to be useful for evaluation of the identification and clonal relationships between Trichophyton rubrum species complex and Trichophyton mentagrophytes species complex isolates. The similarity and diversity of these isolates may be assessed according to different regions by rep-PCR. © 2017 Blackwell Verlag GmbH.

Evaluation of the applicability of amplified rDNA-restriction analysis (ARDRA) to identification of species of the genus Corynebacterium.

PubMed

Vaneechoutte, M; Riegel, P; de Briel, D; Monteil, H; Verschraegen, G; De Rouck, A; Claeys, G

1995-10-01

The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes AluI, CfoI and RsaI was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other: C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction (i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium.

DNA-Based Enzyme Reactors and Systems

PubMed Central

Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.

2016-01-01

During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. PMID:28335267

Subcritical flutter testing and system identification

NASA Technical Reports Server (NTRS)

Houbolt, J. C.

1974-01-01

Treatment is given of system response evaluation, especially in application to subcritical flight and wind tunnel flutter testing of aircraft. An evaluation is made of various existing techniques, in conjuction with a companion survey which reports theoretical and analog experiments made to study the identification of system response characteristics. Various input excitations are considered, and new techniques for analyzing response are explored, particularly in reference to the prevalent practical case where unwanted input noise is present, such as caused by gusts or wind tunnel turbulence. Further developments are also made of system parameter identification techniques.

Decoupling Identification for Serial Two-Link Two-Inertia System

NASA Astrophysics Data System (ADS)

Oaki, Junji; Adachi, Shuichi

The purpose of our study is to develop a precise model by applying the technique of system identification for the model-based control of a nonlinear robot arm, under taking joint-elasticity into consideration. We previously proposed a systematic identification method, called “decoupling identification,” for a “SCARA-type” planar two-link robot arm with elastic joints caused by the Harmonic-drive® reduction gears. The proposed method serves as an extension of the conventional rigid-joint-model-based identification. The robot arm is treated as a serial two-link two-inertia system with nonlinearity. The decoupling identification method using link-accelerometer signals enables the serial two-link two-inertia system to be divided into two linear one-link two-inertia systems. The MATLAB®'s commands for state-space model estimation are utilized in the proposed method. Physical parameters such as motor inertias, link inertias, joint-friction coefficients, and joint-spring coefficients are estimated through the identified one-link two-inertia systems using a gray-box approach. This paper describes accuracy evaluations using the two-link arm for the decoupling identification method under introducing closed-loop-controlled elements and varying amplitude-setup of identification-input. Experimental results show that the identification method also works with closed-loop-controlled elements. Therefore, the identification method is applicable to a “PUMA-type” vertical robot arm under gravity.

DNA Barcodes for Forensically Important Fly Species in Brazil.

PubMed

Koroiva, Ricardo; de Souza, Mirian S; Roque, Fabio de Oliveira; Pepinelli, Mateus

2018-04-07

Here, we analyze 248 DNA barcode sequences of 35 fly species of forensic importance in Brazil. DNA barcoding can be effectively used for specimen identification of these species, allowing the unambiguous identification of 31 species, an overall success rate of 88%. Our results show a high rate of success for molecular identification using DNA barcoding sequences and open new perspectives for immature species identification, a subject on which limited forensic investigations exist in Tropical regions. We also address the implications of building a robust forensic DNA barcode database. A geographic bias is recognized for the COI dataset available for forensically important fly species in Brazil, with concentration of sequences from specimens collected mainly in sites located in the Cerrado, Mata Atlântica, and Pampa biomes.

Chemical Screens Against A Reconstituted Multi-Protein Complex: Myricetin Blocks DnaJ Regulation of DnaK through an Allosteric Mechanism

PubMed Central

Chang, Lyra; Miyata, Yoshinari; Ung, Peter M. U.; Bertelsen, Eric B.; McQuade, Thomas J.; Carlson, Heather A.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.

2011-01-01

SUMMARY DnaK is a molecular chaperone responsible for multiple aspects of proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors, we screened plant-derived extracts against a re-constituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK’s intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems. PMID:21338918

System Identification of a Vortex Lattice Aerodynamic Model

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan; Kholodar, Denis; Dowell, Earl H.

2001-01-01

The state-space presentation of an aerodynamic vortex model is considered from a classical and system identification perspective. Using an aerodynamic vortex model as a numerical simulator of a wing tunnel experiment, both full state and limited state data or measurements are considered. Two possible approaches for system identification are presented and modal controllability and observability are also considered. The theory then is applied to the system identification of a flow over an aerodynamic delta wing and typical results are presented.

[PCR identification of DNA of hosts of the taiga tick nymphs (Ixodes persulcatus: Ixodinae) in St. Petersburg and its suburbs].

PubMed

Grigor'eva, L A; Markov, A V

2011-01-01

PCR identification of host DNA in unfed females and males of taiga tick Ixodes persulcatus was performed. Amplification of each sample was done using primers species-specific by 12S rDNA mitochondrial gene. Four species of small mammals (Apodemus uralensis, Clethrionomys glareolus, Microtus arvalis, and Sorex araneus) and two passeriform bird species (Fringilla coelebs and Parus major) were analysed. For one third of tick samples, hosts of previous stages were established using this method. In five cases, feeding on more than one host species was detected.

A bimodal biometric identification system

NASA Astrophysics Data System (ADS)

Laghari, Mohammad S.; Khuwaja, Gulzar A.

2013-03-01

Biometrics consists of methods for uniquely recognizing humans based upon one or more intrinsic physical or behavioral traits. Physicals are related to the shape of the body. Behavioral are related to the behavior of a person. However, biometric authentication systems suffer from imprecision and difficulty in person recognition due to a number of reasons and no single biometrics is expected to effectively satisfy the requirements of all verification and/or identification applications. Bimodal biometric systems are expected to be more reliable due to the presence of two pieces of evidence and also be able to meet the severe performance requirements imposed by various applications. This paper presents a neural network based bimodal biometric identification system by using human face and handwritten signature features.

Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

PubMed

Kucejová, B; Foury, F

2003-01-01

RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

Multi-level RF identification system

DOEpatents

Steele, Kerry D.; Anderson, Gordon A.; Gilbert, Ronald W.

2004-07-20

A radio frequency identification system having a radio frequency transceiver for generating a continuous wave RF interrogation signal that impinges upon an RF identification tag. An oscillation circuit in the RF identification tag modulates the interrogation signal with a subcarrier of a predetermined frequency and modulates the frequency-modulated signal back to the transmitting interrogator. The interrogator recovers and analyzes the subcarrier signal and determines its frequency. The interrogator generates an output indicative of the frequency of the subcarrier frequency, thereby identifying the responding RFID tag as one of a "class" of RFID tags configured to respond with a subcarrier signal of a predetermined frequency.

Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA.

PubMed

Garcia, Melissa N; Burroughs, Hadley; Gorchakov, Rodion; Gunter, Sarah M; Dumonteil, Eric; Murray, Kristy O; Herrera, Claudia P

2017-04-01

The parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas disease, is widely distributed throughout the Americas, from the southern United States (US) to northern Argentina, and infects at least 6 million people in endemic areas. Much remains unknown about the dynamics of T. cruzi transmission among mammals and triatomine vectors in sylvatic and peridomestic eco-epidemiological cycles, as well as of the risk of transmission to humans in the US. Identification of T. cruzi DTUs among locally-acquired cases is necessary for enhancing our diagnostic and clinical prognostic capacities, as well as to understand parasite transmission cycles. Blood samples from a cohort of 15 confirmed locally-acquired Chagas disease patients from Texas were used for genotyping T. cruzi. Conventional PCR using primers specific for the minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA) confirmed the presence of T. cruzi in 12/15 patients. Genotyping was based on the amplification of the intergenic region of the miniexon gene of T. cruzi and sequencing. Sequences were analyzed by BLAST and phylogenetic analysis by Maximum Likelihood method allowed the identification of non-TcI DTUs infection in six patients, which corresponded to DTUs TcII, TcV or TcVI, but not to TcIII or TcIV. Two of these six patients were also infected with a TcI DTU, indicating mixed infections in those individuals. Electrocardiographic abnormalities were seen among patients with single non-TcI and mixed infections of non-TcI and TcI DTUs. Our results indicate a greater diversity of T. cruzi DTUs circulating among autochthonous human Chagas disease cases in the southern US, including for the first time DTUs from the TcII-TcV-TcVI group. Furthermore, the DTUs infecting human patients in the US are capable of causing Chagasic cardiac disease, highlighting the importance of parasite detection in the population. Copyright © 2017 Elsevier B

The use of Hemastix and the subsequent lack of DNA recovery using the Promega DNA IQ system.

PubMed

Poon, Hiron; Elliott, Jim; Modler, Jeff; Frégeau, Chantal

2009-11-01

Following implementation of our automated process incorporating the Promega DNA IQ system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix chemistries on the DNA IQ system. Our results indicate that one or more chemicals imbedded in the Hemastix reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQ magnetic bead technology. The 3,3',5,5'-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix chemistries on the DNA IQ system forced us to adopt an indirect approach using filter paper to carry out the presumptive test.

Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

PubMed

Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

2016-01-01

Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

DNA-Catalyzed Amide Hydrolysis.

PubMed

Zhou, Cong; Avins, Joshua L; Klauser, Paul C; Brandsen, Benjamin M; Lee, Yujeong; Silverman, Scott K

2016-02-24

DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

PubMed Central

Chen, Y. C.; Eisner, J. D.; Kattar, M. M.; Rassoulian-Barrett, S. L.; LaFe, K.; Yarfitz, S. L.; Limaye, A. P.; Cookson, B. T.

2000-01-01

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by ≤2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of ≥99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species. PMID:10834993

Identification of members of the genera Panaeolus and Psilocybe by a DNA test. A preliminary test for hallucinogenic fungi.

PubMed

Lee, J C; Cole, M; Linacre, A

2000-08-14

Abuse of hallucinogens produced by the fungal genera Psilocybe and Panaeolus are a growing problem. Five species from each of the two genera were examined in this preliminary research and a method that will unambiguously identify fungal samples as being of one of these two genera has been developed. The method uses genus specific DNA sequences within the Internal Transcribed Spacer of the ribosomal gene complex. Amplification of a common DNA product and a genus specific product results in two identifiable products, which facilitates the unambiguous identification of material from these two fungi to generic level.

Identification of metabolically active bacteria in the gut of the generalist Spodoptera littoralis via DNA stable isotope probing using 13C-glucose.

PubMed

Shao, Yongqi; Arias-Cordero, Erika M; Boland, Wilhelm

2013-11-13

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources

PubMed Central

2009-01-01

Background DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. Results We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Conclusion Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org. PMID:19958506

Evaluation of six candidate DNA barcode loci for identification of five important invasive grasses in eastern Australia

PubMed Central

Wang, Aisuo; Gopurenko, David; Wu, Hanwen; Lepschi, Brendan

2017-01-01

Invasive grass weeds reduce farm productivity, threaten biodiversity, and increase weed control costs. Identification of invasive grasses from native grasses has generally relied on the morphological examination of grass floral material. DNA barcoding may provide an alternative means to identify co-occurring native and invasive grasses, particularly during early growth stages when floral characters are unavailable for analysis. However, there are no universal loci available for grass barcoding. We herein evaluated the utility of six candidate loci (atpF intron, matK, ndhK-ndhC, psbE—petL, ETS and ITS) for barcode identification of several economically important invasive grass species frequently found among native grasses in eastern Australia. We evaluated these loci in 66 specimens representing five invasive grass species (Chloris gayana, Eragrostis curvula, Hyparrhenia hirta, Nassella neesiana, Nassella trichotoma) and seven native grass species. Our results indicated that, while no single locus can be universally used as a DNA barcode for distinguishing the grass species examined in this study, two plastid loci (atpF and matK) showed good distinguishing power to separate most of the taxa examined, and could be used as a dual locus to distinguish several of the invasive from the native species. Low PCR success rates were evidenced among two nuclear loci (ETS and ITS), and few species were amplified at these loci, however ETS was able to genetically distinguish the two important invasive Nassella species. Multiple loci analyses also suggested that ETS played a crucial role in allowing identification of the two Nassella species in the multiple loci combinations. PMID:28399170

Evaluation of six candidate DNA barcode loci for identification of five important invasive grasses in eastern Australia.

PubMed

Wang, Aisuo; Gopurenko, David; Wu, Hanwen; Lepschi, Brendan

2017-01-01

Invasive grass weeds reduce farm productivity, threaten biodiversity, and increase weed control costs. Identification of invasive grasses from native grasses has generally relied on the morphological examination of grass floral material. DNA barcoding may provide an alternative means to identify co-occurring native and invasive grasses, particularly during early growth stages when floral characters are unavailable for analysis. However, there are no universal loci available for grass barcoding. We herein evaluated the utility of six candidate loci (atpF intron, matK, ndhK-ndhC, psbE-petL, ETS and ITS) for barcode identification of several economically important invasive grass species frequently found among native grasses in eastern Australia. We evaluated these loci in 66 specimens representing five invasive grass species (Chloris gayana, Eragrostis curvula, Hyparrhenia hirta, Nassella neesiana, Nassella trichotoma) and seven native grass species. Our results indicated that, while no single locus can be universally used as a DNA barcode for distinguishing the grass species examined in this study, two plastid loci (atpF and matK) showed good distinguishing power to separate most of the taxa examined, and could be used as a dual locus to distinguish several of the invasive from the native species. Low PCR success rates were evidenced among two nuclear loci (ETS and ITS), and few species were amplified at these loci, however ETS was able to genetically distinguish the two important invasive Nassella species. Multiple loci analyses also suggested that ETS played a crucial role in allowing identification of the two Nassella species in the multiple loci combinations.

Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences.

PubMed

Kim, W J; Ji, Y; Choi, G; Kang, Y M; Yang, S; Moon, B C

2016-08-05

This study was performed to identify and analyze the phylogenetic relationship among four herbaceous species of the genus Paeonia, P. lactiflora, P. japonica, P. veitchii, and P. suffruticosa, using DNA barcodes. These four species, which are commonly used in traditional medicine as Paeoniae Radix and Moutan Radicis Cortex, are pharmaceutically defined in different ways in the national pharmacopoeias in Korea, Japan, and China. To authenticate the different species used in these medicines, we evaluated rDNA-internal transcribed spacers (ITS), matK and rbcL regions, which provide information capable of effectively distinguishing each species from one another. Seventeen samples were collected from different geographic regions in Korea and China, and DNA barcode regions were amplified using universal primers. Comparative analyses of these DNA barcode sequences revealed species-specific nucleotide sequences capable of discriminating the four Paeonia species. Among the entire sequences of three barcodes, marker nucleotides were identified at three positions in P. lactiflora, eleven in P. japonica, five in P. veitchii, and 25 in P. suffruticosa. Phylogenetic analyses also revealed four distinct clusters showing homogeneous clades with high resolution at the species level. The results demonstrate that the analysis of these three DNA barcode sequences is a reliable method for identifying the four Paeonia species and can be used to authenticate Paeoniae Radix and Moutan Radicis Cortex at the species level. Furthermore, based on the assessment of amplicon sizes, inter/intra-specific distances, marker nucleotides, and phylogenetic analysis, rDNA-ITS was the most suitable DNA barcode for identification of these species.

DNA barcoding of Arctic Ocean holozooplankton for species identification and recognition

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Hopcroft, Russell R.; Kosobokova, Ksenia N.; Nigro, Lisa M.; Ortman, Brian D.; Jennings, Robert M.; Sweetman, Christopher J.

2010-01-01

Zooplankton species diversity and distribution are important measures of environmental change in the Arctic Ocean, and may serve as 'rapid-responders' of climate-induced changes in this fragile ecosystem. The scarcity of taxonomists hampers detailed and up-to-date monitoring of these patterns for the rarer and more problematic species. DNA barcodes (short DNA sequences for species recognition and discovery) provide an alternative approach to accurate identification of known species, and can speed routine analysis of zooplankton samples. During 2004-2008, zooplankton samples were collected during cruises to the central Arctic Ocean and Chukchi Sea. A ˜700 base-pair region of the mitochondrial cytochrome oxidase I (mtCOI) gene was amplified and sequenced for 82 identified specimens of 41 species, including cnidarians (six hydrozoans, one scyphozoan), arthropod crustaceans (five amphipods, 24 copepods, one decapod, and one euphausiid); two chaetognaths; and one nemertean. Phylogenetic analysis used the Neighbor-Joining algorithm with Kimura-2-Parameter (K-2-P) distances, with 1000-fold bootstrapping. K-2-P genetic distances between individuals of the same species ranged from 0.0 to 0.2; genetic distances between species ranged widely from 0.1 to 0.7. The mtCOI gene tree showed monophyly (at 100% bootstrap value) for each of the 26 species for which more than one individual was analyzed. Of seven genera for which more than one species was analyzed, four were shown to be monophyletic; three genera were not resolved. At higher taxonomic levels, only the crustacean order Copepoda was resolved, with bootstrap value of 83%. The mtCOI barcodes accurately discriminated and identified known species of 10 taxonomic groups of Arctic Ocean holozooplankton. A comprehensive DNA barcode database for the estimated 300 described species of Arctic holozooplankton will allow rapid assessment of species diversity and distribution in this climate-vulnerable ocean ecosystem.

Mechanical Strength and Stability of DNA-modified Gold Nanoparticle Systems

NASA Astrophysics Data System (ADS)

Lam, Letisha McLaughlin

Systems in which gold nanoparticles (AuNPs) are functionalized with DNA have the potential for a broad range of applications in gene regulation therapies, drug delivery, sensing, innovative biomaterials and material templates. The use of DNA-modified gold nanoparticle (AuNP-DNA) systems is driven by their ease of assembly with bottom-up methods as well as the tunability of the systems' mechanical, optical, and electronic properties by exploiting AuNP characteristics and behavior in a multi-particle arrangement. Periodic arrangements of AuNPs precisely distributed through ligated DNA linkers may be assembled and used on relatively large length scales, on the order of hundreds of nanometers, for use in potential nanoscale technologies and applications. However, because of the size and heterogeneous composition of AuNP-DNA systems, their stability under mechanical loading is not well understood or quantified on relevant physical scales for these applications. Hence, a large-scale specialized finite-element predictive approach with a dislocation-density based crystalline plasticity has been used to investigate the mechanical stability of AuNP-DNA-ligand systems with AuNPs within the physical dimensions required for plasmon resonance. The crystalline formulation for the AuNPs accounts for multiple crystalline slip, dislocation-density evolution, lattice rotations, and large inelastic strains. A hypoelastic formulation was used for the DNA and the ligands. The nonlinear finite-element scheme is based on accounting for finite elastic and inelastic strains. These approaches were employed to predict and understand the fundamental scale-dependent microstructural behavior, the evolving heterogeneous microstructure, and localized phenomena that can contribute to failure initiation and instability. Each system was loaded using quasi-static plane strain tension and compression to simulate application loading conditions, and the elastic and inelastic evolutions were analyzed for

The application of DNA sequence data for the identification of benthic nematodes from the North Sea

NASA Astrophysics Data System (ADS)

Vogt, Philipp; Miljutina, Maria; Raupach, Michael J.

2014-12-01

Nematodes or roundworms represent one of the most diverse and dominant taxon in marine benthic habitats. Whereas a morphological identification of many species is challenging, the application of molecular markers represents a promising approach for species discrimination and identification. In this study, we used an integrative taxonomic approach, combining both molecular and morphological methods, to characterize nematodes of distinct sex and ontogenetic stages from three sampling sites of the North Sea. Morphospecies were discriminated after first visual determination, followed by a molecular analysis of the nuclear 28S rDNA: D2-D3 marker. By linking each sequence to a morphological voucher, discordant morphological identification was subjected to a so-called reverse taxonomic approach. Molecular operational taxonomic units (MOTUs) and morphospecies were compared for all of the three sampling sites to assess concordance of methodology. In total, 32 MOTUs and 26 morphospecies were assigned, of which 12 taxa were identified as described species. Both approaches showed high concordance in taxon assignment (84.4 %) except for a cluster comprising various Sabatieria species. Our study revealed the high potential of the analyzed fragment as a useful molecular marker for the identification of the North Sea nematodes and highlighted the applicability of this combined taxonomic approach in general.

DNA in the Criminal Justice System: The DNA Success Story in Perspective.

PubMed

Mapes, Anna A; Kloosterman, Ate D; de Poot, Christianne J

2015-07-01

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool. © 2015 American Academy of Forensic Sciences.

iDNA-Prot: Identification of DNA Binding Proteins Using Random Forest with Grey Model

PubMed Central

Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2011-01-01

DNA-binding proteins play crucial roles in various cellular processes. Developing high throughput tools for rapidly and effectively identifying DNA-binding proteins is one of the major challenges in the field of genome annotation. Although many efforts have been made in this regard, further effort is needed to enhance the prediction power. By incorporating the features into the general form of pseudo amino acid composition that were extracted from protein sequences via the “grey model” and by adopting the random forest operation engine, we proposed a new predictor, called iDNA-Prot, for identifying uncharacterized proteins as DNA-binding proteins or non-DNA binding proteins based on their amino acid sequences information alone. The overall success rate by iDNA-Prot was 83.96% that was obtained via jackknife tests on a newly constructed stringent benchmark dataset in which none of the proteins included has pairwise sequence identity to any other in a same subset. In addition to achieving high success rate, the computational time for iDNA-Prot is remarkably shorter in comparison with the relevant existing predictors. Hence it is anticipated that iDNA-Prot may become a useful high throughput tool for large-scale analysis of DNA-binding proteins. As a user-friendly web-server, iDNA-Prot is freely accessible to the public at the web-site on http://icpr.jci.edu.cn/bioinfo/iDNA-Prot or http://www.jci-bioinfo.cn/iDNA-Prot. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results. PMID:21935457

Suitable DNA Barcoding for Identification and Supervision of Piper kadsura in Chinese Medicine Markets.

PubMed

Yu, Ning; Gu, Hong; Wei, Yulong; Zhu, Ning; Wang, Yanli; Zhang, Haiping; Zhu, Yue; Zhang, Xin; Ma, Chao; Sun, Aidong

2016-09-12

Piper kadsura is a vine-like medicinal plant which is widely used in clinical treatment. However, P. kadsura is often substituted by other materials in the markets, thereby causing health risks. In this study, 38 P. kadsura samples and eight sequences from GenBank, including a closely-related species and common adulterants were collected. This study aimed to identify an effective DNA barcode from four popular DNA loci for P. kadsura authentication. The success rates of PCR amplification, sequencing, and sequence acquisition of matK were 10.5%, 75%, and 7.9%, respectively; for rbcL they were 89.5%, 8.8%, and 7.9%, respectively; ITS2 rates were 86.8%, 3.0%, and 2.6%, respectively, while for psbA-trnH they were all 100%, which is much higher than for the other three loci. The sequences were aligned using Muscle, genetic distances were computed using MEGA 5.2.2, and barcoding gap was performed using TAXON DNA. Phylogenetic analysis showed that psbA-trnH could clearly distinguish P. kadsura from its closely related species and the common adulterant. psbA-trnH was then used to evaluate the fake proportions of P. kadsura. Results showed that 18.4% of P. kadsura samples were fake, indicating that adulterant species exist in the Chinese markets. Two-dimensional DNA barcoding imaging of P. kadsura was conducted, which was beneficial to the management of P. kadsura. We conclude that the psbA-trnH region is a powerful tool for P. kadsura identification and supervision in the current medicine markets.

IDENTIFICATION OF STEREOCHEMICAL CONFIGURATIONS OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8 CELLS

EPA Science Inventory

Identification of Sterochemical Configurations of Cyclopent A[cd]Pyrene DNA Adducts in Strain A/J Mouse Lung and C3H10T1/2CL8 Cells.

Four major and several minor DNA adducts were resolved by 32P-postlabeling analysis of DNA from strain A/J mouse lung and C3H10T1/2CL8 (C3H...

Evaluation of the utility of a glycemic pattern identification system.

PubMed

Otto, Erik A; Tannan, Vinay

2014-07-01

With the increasing prevalence of systems allowing automated, real-time transmission of blood glucose data there is a need for pattern recognition techniques that can inform of deleterious patterns in glycemic control when people test. We evaluated the utility of pattern identification with a novel pattern identification system named Vigilant™ and compared it to standard pattern identification methods in diabetes. To characterize the importance of an identified pattern we evaluated the relative risk of future hypoglycemic and hyperglycemic events in diurnal periods following identification of a pattern in a data set of 536 patients with diabetes. We evaluated events 2 days, 7 days, 30 days, and 61-90 days from pattern identification, across diabetes types and cohorts of glycemic control, and also compared the system to 6 pattern identification methods consisting of deleterious event counts and percentages over 5-, 14-, and 30-day windows. Episodes of hypoglycemia, hyperglycemia, severe hypoglycemia, and severe hyperglycemia were 120%, 46%, 123%, and 76% more likely after pattern identification, respectively, compared to periods when no pattern was identified. The system was also significantly more predictive of deleterious events than other pattern identification methods evaluated, and was persistently predictive up to 3 months after pattern identification. The system identified patterns that are significantly predictive of deleterious glycemic events, and more so relative to many pattern identification methods used in diabetes management today. Further study will inform how improved pattern identification can lead to improved glycemic control. © 2014 Diabetes Technology Society.

A General Method for Discovering Inhibitors of Protein–DNA Interactions Using Photonic Crystal Biosensors

PubMed Central

Chan, Leo L.; Pineda, Maria; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2009-01-01

Protein–DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein–DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein–DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein–DNA binding. The PC technology is used to detect binding between protein–DNA interactions that are DNA-sequence-dependent (the bacterial toxin–antitoxin system MazEF) and those that are DNA-sequence-independent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF–DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein–DNA binding. PMID:18582039

In-Flight System Identification

NASA Technical Reports Server (NTRS)

Morelli, Eugene A.

1998-01-01

A method is proposed and studied whereby the system identification cycle consisting of experiment design and data analysis can be repeatedly implemented aboard a test aircraft in real time. This adaptive in-flight system identification scheme has many advantages, including increased flight test efficiency, adaptability to dynamic characteristics that are imperfectly known a priori, in-flight improvement of data quality through iterative input design, and immediate feedback of the quality of flight test results. The technique uses equation error in the frequency domain with a recursive Fourier transform for the real time data analysis, and simple design methods employing square wave input forms to design the test inputs in flight. Simulation examples are used to demonstrate that the technique produces increasingly accurate model parameter estimates resulting from sequentially designed and implemented flight test maneuvers. The method has reasonable computational requirements, and could be implemented aboard an aircraft in real time.

Anti-DNA Ig peptides promote Treg cell activity in systemic lupus erythematosus patients.

PubMed

Hahn, Bevra H; Anderson, Marissa; Le, Elizabeth; La Cava, Antonio

2008-08-01

Treg cells oppose autoreactive responses in several autoimmune diseases, and their frequency is reduced in systemic lupus erythematosus (SLE). In murine lupus models, treatment with anti-DNA Ig-based peptides can expand the number of Treg cells in vivo. This study was undertaken to test the possibility that functional human Treg cells can be induced by exposure to anti-DNA Ig-based peptides. Peripheral blood mononuclear cells were isolated from 36 lupus patients and 32 healthy individuals matched for ethnicity, sex, and age. Short-term culture experiments in the presence of several independent stimuli including anti-DNA Ig peptides were followed by flow cytometric analysis for identification of CD4+,CD25(high) T cells, cell sorting for in vitro suppression assays, and analysis of correlations between the expression of forkhead box P3 (FoxP3) and serologic and clinical characteristics of the SLE patients. The number of in vitro CD4+,CD25(high) T cells increased after culture with anti-DNA Ig peptides in the SLE patients, but not in the controls. The expanded CD4+,CD25(high) T cells required FoxP3 for cell contact-mediated suppression of proliferation and interferon-gamma production in target CD4+,CD25- T cells. The induction of FoxP3 in SLE Treg cells occurred only in seropositive patients, and was correlated with anti-DNA and IgG serum titers. These results suggest a new modality to reverse the functional deficit of Treg cells in SLE patients with positive autoimmune serology, and identify a new strategy to enhance immunoregulatory T cell activity in human SLE.

Iterated function systems for DNA replication

NASA Astrophysics Data System (ADS)

Gaspard, Pierre

2017-10-01

The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

Nanofluidic Lab-On-Chip Technology for DNA Identification

DTIC Science & Technology

2013-09-30

samples Fluorescently labeled (FAM tag) DNA oligomers (10, 20, and 50 bases long) were purchased with standard desalting and additional HPLC purification...A.2 DNA samples: DNA oligomers (10, 20, 50 nt long) were purchased with standard desalting and additional HPLC purification for the 50 base

Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe

PubMed Central

Dincă, Vlad; Zakharov, Evgeny V.; Hebert, Paul D. N.; Vila, Roger

2011-01-01

DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development. PMID:20702462

Application of identification techniques to remote manipulator system flight data

NASA Technical Reports Server (NTRS)

Shepard, G. D.; Lepanto, J. A.; Metzinger, R. W.; Fogel, E.

1983-01-01

This paper addresses the application of identification techniques to flight data from the Space Shuttle Remote Manipulator System (RMS). A description of the remote manipulator, including structural and control system characteristics, sensors, and actuators is given. A brief overview of system identification procedures is presented, and the practical aspects of implementing system identification algorithms are discussed. In particular, the problems posed by desampling rate, numerical error, and system nonlinearities are considered. Simulation predictions of damping, frequency, and system order are compared with values identified from flight data to support an evaluation of RMS structural and control system models. Finally, conclusions are drawn regarding the application of identification techniques to flight data obtained from a flexible space structure.

Anti-collision radio-frequency identification system using passive SAW tags

NASA Astrophysics Data System (ADS)

Sorokin, A. V.; Shepeta, A. P.

2017-06-01

Modern multi sensor systems should have high operating speed and resistance to climate impacts. Radiofrequency systems use passive SAW tags for identification items and vehicles. These tags find application in industry, traffic remote control systems, and railway remote traffic control systems for identification and speed measuring. However, collision of the passive SAW RFID tags hinders development passive RFID SAW technology in Industry. The collision problem for passive SAW tags leads for incorrect identification and encoding each tag. In our researching, we suggest approach for identification of several passive SAW tags in collision case.

Geographic origin and individual assignment of Shorea platyclados (Dipterocarpaceae) for forensic identification

PubMed Central

Diway, Bibian; Khoo, Eyen

2017-01-01

The development of timber tracking methods based on genetic markers can provide scientific evidence to verify the origin of timber products and fulfill the growing requirement for sustainable forestry practices. In this study, the origin of an important Dark Red Meranti wood, Shorea platyclados, was studied by using the combination of seven chloroplast DNA and 15 short tandem repeats (STRs) markers. A total of 27 natural populations of S. platyclados were sampled throughout Malaysia to establish population level and individual level identification databases. A haplotype map was generated from chloroplast DNA sequencing for population identification, resulting in 29 multilocus haplotypes, based on 39 informative intraspecific variable sites. Subsequently, a DNA profiling database was developed from 15 STRs allowing for individual identification in Malaysia. Cluster analysis divided the 27 populations into two genetic clusters, corresponding to the region of Eastern and Western Malaysia. The conservativeness tests showed that the Malaysia database is conservative after removal of bias from population subdivision and sampling effects. Independent self-assignment tests correctly assigned individuals to the database in an overall 60.60−94.95% of cases for identified populations, and in 98.99−99.23% of cases for identified regions. Both the chloroplast DNA database and the STRs appear to be useful for tracking timber originating in Malaysia. Hence, this DNA-based method could serve as an effective addition tool to the existing forensic timber identification system for ensuring the sustainably management of this species into the future. PMID:28430826

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates

PubMed Central

Faron, Matthew L.; Buchan, Blake W.; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L.; Granato, Paul A.; Wilson, Deborah A.; Procop, Gary W.; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A.

2015-01-01

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria. PMID:26529504

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

PubMed

Faron, Matthew L; Buchan, Blake W; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L; Granato, Paul A; Wilson, Deborah A; Procop, Gary W; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A

2015-01-01

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

Validation of a rapid DNA process with the RapidHIT® ID system using GlobalFiler® Express chemistry, a platform optimized for decentralized testing environments.

PubMed

Salceda, Susana; Barican, Arnaldo; Buscaino, Jacklyn; Goldman, Bruce; Klevenberg, Jim; Kuhn, Melissa; Lehto, Dennis; Lin, Frank; Nguyen, Phong; Park, Charles; Pearson, Francesca; Pittaro, Rick; Salodkar, Sayali; Schueren, Robert; Smith, Corey; Troup, Charles; Tsou, Dean; Vangbo, Mattias; Wunderle, Justus; King, David

2017-05-01

The RapidHIT ® ID is a fully automated sample-to-answer system for short tandem repeat (STR)-based human identification. The RapidHIT ID has been optimized for use in decentralized environments and processes presumed single source DNA samples, generating Combined DNA Index System (CODIS)-compatible DNA profiles in less than 90min. The system is easy to use, requiring less than one minute of hands-on time. Profiles are reviewed using centralized linking software, RapidLINK™ (IntegenX, Pleasanton, CA), a software tool designed to collate DNA profiles from single or multiple RapidHIT ID systems at different geographic locations. The RapidHIT ID has been designed to employ GlobalFiler ® Express and AmpFLSTR ® NGMSElect™, Thermo Fisher Scientific (Waltham, MA) STR chemistries. The Developmental Validation studies were performed using GlobalFiler ® Express with single source reference samples according to Scientific Working Group for DNA Analysis Methods guidelines. These results show that multiple RapidHIT ID systems networked with RapidLINK software form a highly reliable system for wide-scale deployment in locations such as police booking stations and border crossings enabling real-time testing of arrestees, potential human trafficking victims, and other instances where rapid turnaround is essential. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

DNA barcode and identification of the varieties and provenances of Taiwan's domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences.

PubMed

Lee, Shih-Chieh; Wang, Chia-Hsiang; Yen, Cheng-En; Chang, Chieh

2017-04-01

The major aim of made tea identification is to identify the variety and provenance of the tea plant. The present experiment used 113 tea plants [Camellia sinensis (L.) O. Kuntze] housed at the Tea Research and Extension Substation, from which 113 internal transcribed spacer 2 (ITS2) fragments, 104 trnL intron, and 98 trnL-trnF intergenic sequence region DNA sequences were successfully sequenced. The similarity of the ITS2 nucleotide sequences between tea plants housed at the Tea Research and Extension Substation was 0.379-0.994. In this polymerase chain reaction-amplified noncoding region, no varieties possessed identical sequences. Compared with the trnL intron and trnL-trnF intergenic sequence fragments of chloroplast cpDNA, the proportion of ITS2 nucleotide sequence variation was large and is more suitable for establishing a DNA barcode database to identify tea plant varieties. After establishing the database, 30 imported teas and 35 domestic made teas were used in this model system to explore the feasibility of using ITS2 sequences to identify the varieties and provenances of made teas. A phylogenetic tree was constructed using ITS2 sequences with the unweighted pair group method with arithmetic mean, which indicated that the same variety of tea plant is likely to be successfully categorized into one cluster, but contamination from other tea plants was also detected. This result provides molecular evidence that the similarity between important tea varieties in Taiwan remains high. We suggest a direct, wide collection of made tea and original samples of tea plants to establish an ITS2 sequence molecular barcode identification database to identify the varieties and provenances of tea plants. The DNA barcode comparison method can satisfy the need for a rapid, low-cost, frontline differentiation of the large amount of made teas from Taiwan and abroad, and can provide molecular evidence of their varieties and provenances. Copyright © 2016. Published by Elsevier B.V.

Building a DNA barcode library of Alaska's non-marine arthropods.

PubMed

Sikes, Derek S; Bowser, Matthew; Morton, John M; Bickford, Casey; Meierotto, Sarah; Hildebrandt, Kyndall

2017-03-01

Climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Alaska has a limited taxonomic workforce to address these changes. We are building a DNA barcode library to facilitate a metabarcoding approach to monitoring non-marine arthropods. Working with the Canadian Centre for DNA Barcoding, we obtained DNA barcodes from recently collected and authoritatively identified specimens in the University of Alaska Museum (UAM) Insect Collection and the Kenai National Wildlife Refuge collection. We submitted tissues from 4776 specimens, of which 81% yielded DNA barcodes representing 1662 species and 1788 Barcode Index Numbers (BINs), of primarily terrestrial, large-bodied arthropods. This represents 84% of the species available for DNA barcoding in the UAM Insect Collection. There are now 4020 Alaskan arthropod species represented by DNA barcodes, after including all records in Barcode of Life Data Systems (BOLD) of species that occur in Alaska - i.e., 48.5% of the 8277 Alaskan, non-marine-arthropod, named species have associated DNA barcodes. An assessment of the identification power of the library in its current state yielded fewer species-level identifications than expected, but the results were not discouraging. We believe we are the first to deliberately begin development of a DNA barcode library of the entire arthropod fauna for a North American state or province. Although far from complete, this library will become increasingly valuable as more species are added and costs to obtain DNA sequences fall.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

PubMed Central

Haddad, Diana; Bilcikova, Erika; Witney, Adam A.; Carlton, Jane M.; White, Charles E.; Blair, Peter L.; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C.; Carucci, Daniel J.; Weiss, Walter R.

2004-01-01

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens. PMID:14977966

Substructure System Identification for Finite Element Model Updating

NASA Technical Reports Server (NTRS)

Craig, Roy R., Jr.; Blades, Eric L.

1997-01-01

This report summarizes research conducted under a NASA grant on the topic 'Substructure System Identification for Finite Element Model Updating.' The research concerns ongoing development of the Substructure System Identification Algorithm (SSID Algorithm), a system identification algorithm that can be used to obtain mathematical models of substructures, like Space Shuttle payloads. In the present study, particular attention was given to the following topics: making the algorithm robust to noisy test data, extending the algorithm to accept experimental FRF data that covers a broad frequency bandwidth, and developing a test analytical model (TAM) for use in relating test data to reduced-order finite element models.

System identification of jet engines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugiyama, N.

2000-01-01

System identification plays an important role in advanced control systems for jet engines, in which controls are performed adaptively using data from the actual engine and the identified engine. An identification technique for jet engine using the Constant Gain Extended Kalman Filter (CGEKF) is described. The filter is constructed for a two-spool turbofan engine. The CGEKF filter developed here can recognize parameter change in engine components and estimate unmeasurable variables over whole flight conditions. These capabilities are useful for an advanced Full Authority Digital Electric Control (FADEC). Effects of measurement noise and bias, effects of operating point and unpredicted performancemore » change are discussed. Some experimental results using the actual engine are shown to evaluate the effectiveness of CGEKF filter.« less

DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters

NASA Astrophysics Data System (ADS)

Fernández-Álvarez, Fernando Ángel; Machordom, Annie

2013-09-01

For several groups, like nemerteans, morphology-based identification is a hard discipline, but DNA barcoding may help non-experts in the identification process. In this study, DNA barcoding is used to reveal the cryptic invasion of Pacific Cephalothrix cf. simula into Atlantic and Mediterranean coasts. Although DNA barcoding is a promising method for the identification of Nemertea, only 6 % of the known number of nemertean species is currently associated with a correct DNA barcode. Therefore, additional morphological and molecular studies are necessary to advance the utility of DNA barcoding in the characterisation of possible nemertean alien invasions.

Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila.

PubMed

Żak, Mariusz; Zaborowski, Piotr; Baczewska-Rej, Milena; Zasada, Aleksandra A; Matuszewska, Renata; Krogulska, Bożena

2011-12-20

For the last five years, Legionella sp. infections and legionnaire's disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations. The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected. We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain. The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

PubMed

Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

2014-05-01

Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

DNA synthesis inhibitors for the treatment of gastrointestinal cancer.

PubMed

Yasui, Hiroshi; Tsurita, Giichiro; Imai, Kohzoh

2014-11-01

Intensive laboratory, preclinical and clinical studies have identified and validated molecular targets in cancers, leading to a shift toward the development of novel, rationally designed and specific therapeutic agents. However, gastrointestinal cancers continue to have a poor prognosis, largely due to drug resistance. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies. Conventional agents, including DNA synthesis inhibitors such as fluoropyrimidines and platinum analogs, remain the most effective therapeutics and are the standards against which new drugs are compared. Novel DNA synthesis inhibitors for the treatment of gastrointestinal malignancies include a combination of the antimetabolite TAS-102, which consists of trifluorothymidine with a thymidine phosphorylase inhibitor, and a novel micellar formulation of cisplatin NC-6004 that uses a nanotechnology-based drug delivery system. The challenges of translational cancer research using DNA synthesis inhibitors include the identification of drugs that are specific to tumor cells to reduce toxicity and increase antitumor efficacy, biomarkers to predict pharmacological responses to chemotherapeutic drugs, identification of ways to overcome drug resistance and development of novel combination therapies with DNA synthesis inhibitors and other cancer therapies, such as targeted molecular therapeutics. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies.

Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

NASA Astrophysics Data System (ADS)

Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

2016-04-01

Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

PubMed Central

Vivas, Caio Vinicius; Moraes, Ramiris César Souza; Alves-Araújo, Anderson; Alves, Marccus; Mariano-Neto, Eduardo; van den Berg, Cássio; Gaiotto, Fernanda Amato

2014-01-01

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest. PMID:25505841

Pioneer identification of fake tiger claws using morphometric and DNA-based analysis in wildlife forensics in India.

PubMed

Vipin; Sharma, Vinita; Sharma, Chandra Prakash; Kumar, Ved Prakash; Goyal, Surendra Prakash

2016-09-01

The illegal trade in wildlife is a serious threat to the existence of wild animals throughout the world. The short supply and high demand for wildlife articles have caused an influx of many different forms of fake wildlife articles into this trade. The task of identifying the materials used in making such articles poses challenges in wildlife forensics as different approaches are required for species identification. Claws constitute 3.8% of the illegal animal parts (n=2899) received at the Wildlife Institute of India (WII) for species identification. We describe the identification of seized suspected tiger claws (n=18) using a combined approach of morphometric and DNA-based analysis. The differential keratin density, determined using X-ray radiographs, indicated that none of the 18 claws were of any large cat but were fake. We determined three claw measurements, viz. ac (from the external coronary dermo-epidermal interface to the epidermis of the skin fold connecting the palmar flanges of the coronary horn), bc (from the claw tip to the epidermis of the skin fold connecting the palmar flanges of the coronary horn) and the ratio bc/ac, for all the seized (n=18), tiger (n=23) and leopard (n=49) claws. Univariate and multivariate statistical analyses were performed using SPSS. A scatter plot generated using canonical discriminant function analysis revealed that of the 18 seized claws, 14 claws formed a cluster separate from the clusters of the tiger and leopard claws, whereas the remaining four claws were within the leopard cluster. Because a discrepancy was observed between the X-ray images and the measurements of these four claws, one of the claw that clustered with the leopard claws was chosen randomly and DNA analysis carried out using the cyt b (137bp) and 16S rRNA (410bp) genes. A BLAST search and comparison with the reference database at WII indicated that the keratin material of the claw was derived from Bos taurus (cattle). This is a pioneering discovery, and

Fractional System Identification: An Approach Using Continuous Order-Distributions

NASA Technical Reports Server (NTRS)

Hartley, Tom T.; Lorenzo, Carl F.

1999-01-01

This paper discusses the identification of fractional- and integer-order systems using the concept of continuous order-distribution. Based on the ability to define systems using continuous order-distributions, it is shown that frequency domain system identification can be performed using least squares techniques after discretizing the order-distribution.

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

NASA Astrophysics Data System (ADS)

Lee, Chia-Yen; Lee, Gwo-Bin; Lin, Jr-Lung; Huang, Fu-Chun; Liao, Chia-Sheng

2005-06-01

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations.

PubMed

Zhou, Changqing; Kandemir, Irfan; Walsh, Douglas B; Zalom, Frank G; Lavine, Laura Corley

2012-01-01

The western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification. This study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of F(st) and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States. This study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.

Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

PubMed

El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

2013-07-01

Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks. Copyright © 2013. Published by Elsevier Ltd.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

PubMed Central

Pérez-Osorio, Ailyn C.; Boyle, David S.; Ingham, Zachary K.; Ostash, Alla; Gautom, Romesh K.; Colombel, Craig; Houze, Yolanda

2012-01-01

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampinr TB identification; 60.6% and 100% for isoniazidr TB identification; and 75.0% and 98.1% for pyrazinamider TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced. PMID:22162548

Classification of Sharks in the Egyptian Mediterranean Waters Using Morphological and DNA Barcoding Approaches

PubMed Central

Moftah, Marie; Abdel Aziz, Sayeda H.; Elramah, Sara; Favereaux, Alexandre

2011-01-01

The identification of species constitutes the first basic step in phylogenetic studies, biodiversity monitoring and conservation. DNA barcoding, i.e. the sequencing of a short standardized region of DNA, has been proposed as a new tool for animal species identification. The present study provides an update on the composition of shark in the Egyptian Mediterranean waters off Alexandria, since the latest study to date was performed 30 years ago, DNA barcoding was used in addition to classical taxonomical methodologies. Thus, 51 specimen were DNA barcoded for a 667 bp region of the mitochondrial COI gene. Although DNA barcoding aims at developing species identification systems, some phylogenetic signals were apparent in the data. In the neighbor-joining tree, 8 major clusters were apparent, each of them containing individuals belonging to the same species, and most with 100% bootstrap value. This study is the first to our knowledge to use DNA barcoding of the mitochondrial COI gene in order to confirm the presence of species Squalus acanthias, Oxynotus centrina, Squatina squatina, Scyliorhinus canicula, Scyliorhinus stellaris, Mustelus mustelus, Mustelus punctulatus and Carcharhinus altimus in the Egyptian Mediterranean waters. Finally, our study is the starting point of a new barcoding database concerning shark composition in the Egyptian Mediterranean waters (Barcoding of Egyptian Mediterranean Sharks [BEMS], http://www.boldsystems.org/views/projectlist.php?&#Barcoding%20Fish%20%28FishBOL%29). PMID:22087242

[DNA Extraction from Old Bones by AutoMate Express™ System].

PubMed

Li, B; Lü, Z

2017-08-01

To establish a method for extracting DNA from old bones by AutoMate Express™ system. Bones were grinded into powder by freeze-mill. After extraction by AutoMate Express™, DNA were amplified and genotyped by Identifiler®Plus and MinFiler™ kits. DNA were extracted from 10 old bone samples, which kept in different environments with the postmortem interval from 10 to 20 years, in 3 hours by AutoMate Express™ system. Complete STR typing results were obtained from 8 samples. AutoMate Express™ system can quickly and efficiently extract DNA from old bones, which can be applied in forensic practice. Copyright© by the Editorial Department of Journal of Forensic Medicine

Molecular identification of vertebrate and hemoparasite DNA within mosquito blood meals from eastern North Dakota.

PubMed

Mehus, Joseph O; Vaughan, Jefferson A

2013-11-01

To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of vector

Molecular Identification of Vertebrate and Hemoparasite DNA Within Mosquito Blood Meals From Eastern North Dakota

PubMed Central

Vaughan, Jefferson A.

2013-01-01

Abstract To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of

Input-output identification of controlled discrete manufacturing systems

NASA Astrophysics Data System (ADS)

Estrada-Vargas, Ana Paula; López-Mellado, Ernesto; Lesage, Jean-Jacques

2014-03-01

The automated construction of discrete event models from observations of external system's behaviour is addressed. This problem, often referred to as system identification, allows obtaining models of ill-known (or even unknown) systems. In this article, an identification method for discrete event systems (DESs) controlled by a programmable logic controller is presented. The method allows processing a large quantity of observed long sequences of input/output signals generated by the controller and yields an interpreted Petri net model describing the closed-loop behaviour of the automated DESs. The proposed technique allows the identification of actual complex systems because it is sufficiently efficient and well adapted to cope with both the technological characteristics of industrial controllers and data collection requirements. Based on polynomial-time algorithms, the method is implemented as an efficient software tool which constructs and draws the model automatically; an overview of this tool is given through a case study dealing with an automated manufacturing system.

The effectiveness of three regions in mitochondrial genome for aphid DNA barcoding: a case in Lachininae.

PubMed

Chen, Rui; Jiang, Li-Yun; Qiao, Ge-Xia

2012-01-01

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding. Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in "best match" and 90.8% in "best close match") and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of "tag barcodes" is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the "barcoding overlap" can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the "best close match" technique. A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of "tag barcodes" can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.

System Identification Methods for Aircraft Flight Control Development and Validation

DOT National Transportation Integrated Search

1995-10-01

System-identification methods compose a mathematical model, or series of models, : from measurements of inputs and outputs of dynamic systems. This paper : discusses the use of frequency-domain system-identification methods for the : development and ...

DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

PubMed

Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

2012-01-01

DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

Rotorcraft System Identification (L’Identification des Systemes de Voilures ;Tournantes)

DTIC Science & Technology

1991-09-01

Icgroupe detraivail No.18 sur t~idenrifleariisndes systtmes tie vcilures trturnatnt~s. a proe~dde lapplicatioindte toure la gamme tic mdtboties...AD-A244 250 -liii ii _,AGARD-AR-280 , AGAL?1Mw ADVISORY GROUP FOR AEROSPACE RESEARCH & DEVELOPMENT ag 7 RUE ANCELLE 92200 NEUILLY SUR SEINE FRANCE...DEVELOPMENT 7 RUE ANCELLE 92200 NEUILLY SUR SEINE FRANCE AGARD ADVISORY REPORT 280 Rotorcraft System Identification (L’ldcntification des Syst~mes de Voidures

49 CFR 1544.231 - Airport-approved and exclusive area personnel identification systems.

Code of Federal Regulations, 2010 CFR

2010-10-01

... carry out a personnel identification system for identification media that are airport-approved, or identification media that are issued for use in an exclusive area. The system must include the following: (1) Personnel identification media that— (i) Convey a full face image, full name, employer, and identification...

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.